High-throughput nucleic acid testing of biological samples

Abstract

The subject matter disclosed herein relates to methods for rapid, sensitive, and high-throughput nucleic acid testing of biological samples, such as blood, serum, or plasma samples from a donor, as well as systems capable of performing such high-throughput nucleic acid testing.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application is a continuation of U.S. Provisional Patent Application No. 63 / 302,957, filed January 25, 2022, U.S. Provisional Patent Application No. 63 / 302,959, filed January 25, 2022, and U.S. Provisional Patent Application No. 63 / 302,982, filed January 25, 2022, and U.S. Provisional Patent Application No. 63 / 302,939, filed January 25, 2022, and U.S. Provisional Patent Application No. 63 / 302,940, filed April 29, 2021. No. 63 / 181,799, filed April 29, 2021, and U.S. Provisional Patent Application No. 63 / 181,822, filed April 29, 2021, and U.S. Provisional Patent Application No. 63 / 181,874, filed April 29, 2021, and U.S. Provisional Patent Application No. 63 / 181,880, filed April 29, 2021, the entire contents of each of which are incorporated herein by reference. [Background technology]

[0002] Areas of disclosure The subject matter disclosed herein relates to methods for rapid, sensitive, and high-throughput nucleic acid testing of biological samples, such as blood, serum, or plasma samples from donors and / or patients, as well as systems capable of performing such high-throughput nucleic acid testing.

[0003] 2. Description of Related Art Screening of donated blood and plasma plays a critical role in protecting the supply of lifesaving whole blood, plasma, platelets, red blood cells, and blood products manufactured from whole blood or plasma. Not surprisingly, screening of donated blood and plasma is often highly regulated to ensure that the blood and plasma are free of pathogens and infectious agents. Government agencies and other accredited organizations provide detailed guidance on their jurisdiction's whole blood and plasma screening processes. For example, the U.S. Food and Drug Administration provides guidance in the United States on appropriate donated blood screening processes. Other organizations, such as the World Health Organization (WHO) or specific national health organizations, also provide guidance on donated blood screening. While common approaches exist for screening donated blood across various jurisdictions, no universal requirements exist, and screening guidelines vary from jurisdiction to jurisdiction.

[0004] Detection of pathogens and infectious agents in the donated blood supply currently often involves serology and nucleic acid testing ("NAT"). Serology refers to immunoassay testing to detect the presence of antigens from a pathogen or infectious agent and / or antibodies produced by the donor against the pathogen or infectious agent. In contrast, NAT refers to the detection of genetic material (e.g., DNA and / or RNA) associated with a pathogen or infectious agent. In an exemplary workflow for screening donated blood for transfusion, blood is collected from the donor at a collection site into blood bags and multiple sample collection tubes. The donor also completes a survey requesting information about the donated blood. The blood bags are transported to a blood collection facility for further processing and storage. The collection tubes are transported to a blood screening laboratory where the sample is subjected to testing for pathogens or infectious diseases and blood typing and determination. Samples may be subjected to immunoassay testing for antiviral antibodies, such as human immunodeficiency virus (HIV), hepatitis B virus (HBV)-specific antigens, e.g., hepatitis B surface antigen (HBsAg), human T-cell lymphotropic virus (HTLV), Chagas, cytomegalovirus (CMV), syphilis, anti-HBc, and anti-HCV antibodies. Samples may also be subjected to NAT for, e.g., HIV, HCV, HBV, Babesia, Zika, and West Nile virus. Generally, the informatics system tracks the test results for each test on each sample. If an analyte test result is positive, the donated blood is quarantined and the donor is notified of the screening results. If all tests are negative for a particular sample, the donated blood is shipped for clinical use.

[0005] Similarly, in an exemplary workflow for screening donated plasma, plasma is collected from a donor, such as through plasmapheresis. During this process, whole blood is withdrawn, plasma is collected, and the remaining blood components are returned to the donor. Plasma screening may include the immunoassays and NATs described above, and may also include NATs for parvovirus B19 and hepatitis A virus (HAV). While the above tests are commonly used for screening, different jurisdictions or as new outbreaks and infections occur may use different tests for new analytes or targets of interest.

[0006] In many jurisdictions, NATs are required due to their greater sensitivity in detecting low levels of pathogens that may be below the detection limit of serological assays. Jurisdictions often rely on a duplicate testing strategy, whereby donated blood is screened by a low-cost, high-throughput serological assay to determine whether the donor has been infected (or is currently infected) with a transfusion-transmitted pathogen or infectious agent, and then by the time-consuming, more direct process of NAT to determine whether they are currently infected.

[0007] However, current NAT-based screening involves test times that can be several hours longer than serology testing. Due to relatively high costs and long turnaround times, NAT-based screening of donated blood and plasma creates significant bottlenecks and costs in whole blood and plasma screening. The time difference between the time of whole blood or plasma donation and the time it takes for the donated whole blood or plasma to be shipped becomes particularly important during national / global health emergencies. Additionally, the long time it takes for whole blood to be shipped shortens the shelf life of whole blood and its components, such as platelets and red blood cells, which can be fractionated from whole blood. Attempts have been made to blunt the impact of the significant time investment in NAT-based screening by optimizing and implementing workflow strategies. These techniques include pooling and personnel scheduling to ensure efficiency despite the complex cadence of current NAT-based screening. However, each of these workflow strategies presents its own challenges. Personnel scheduling for NAT-based screening can be costly. With regard to pooling in particular, complex sample pooling strategies have been developed to facilitate higher throughput. Furthermore, traditional pooling requires additional time and equipment for pooling, and adds time and expense associated with disassembling the pooled samples if a pathogen or infectious agent is detected in the pooled samples. There is also a high risk of errors introduced by poor quality procedures during pool preparation, when recording the individual samples in each pool, and when subsequently rescreening the pooled samples to identify individual samples that are positive for the tested pathogen or infectious agent. These drawbacks of pooling are exacerbated in certain situations, such as plasma centers, where large-scale pooling is the industry standard and disassembling and rescreening of large pools may be necessary.

[0008] Furthermore, despite the relatively low cost and rapid turnaround time of serological screening, the labor and infrastructure costs associated with implementing parallel screening systems present additional logistical challenges, especially in low-resource areas. Current parallel screening systems are expensive and time-consuming, leading to the introduction of various resource-intensive solutions. NAT-based screening is often centralized to take advantage of economies of scale. However, these solutions are not without drawbacks, including the cost and time required to transport samples to a central screening laboratory located far from blood collection, processing, and storage facilities, further slowing down the shipping process.

[0009] Therefore, there is a need for NATs that can be performed at higher throughput, including NATs that require faster turnaround times than currently available NATs for whole blood and plasma screening. There is also a need for whole blood and plasma screening assays that can replace current processes that require both serology and NAT. The present invention meets these and other needs. Summary of the Invention

[0010] The present disclosure provides a method for screening a sample of donor blood for shipping the donor blood or material from the donor for clinical use.

[0011] The present disclosure provides a method for screening one or more samples of donor blood for shipping the donor blood or material from a donor for clinical use, the method comprising performing nucleic acid analysis on the one or more samples of donor blood to detect a plurality of pathogens or infectious agents, wherein a predetermined level of nucleic acid from each of the plurality of pathogens or infectious agents based on the nucleic acid analysis is determined to indicate shipping the donor blood or donor material for clinical use, wherein shipping the donor blood for clinical use occurs in about 15 to about 60 minutes, e.g., about 20 to about 60 minutes, from the initial drawing of the one or more samples for performing the nucleic acid analysis.

[0012] The present disclosure also provides a method for screening one or more samples of donor blood for shipment of the donor blood or material from a donor for clinical use, the method comprising performing nucleic acid analysis on the samples of donor blood to detect a plurality of pathogens or infectious agents, wherein a predetermined level of nucleic acid from each of the plurality of pathogens or infectious agents based on the nucleic acid analysis is determined, indicating shipment of the donor blood for clinical use, the method comprising screening a plurality of samples of donor blood for shipment of the donor blood or material for clinical use, wherein the determination based on the nucleic acid analysis is performed within about 20 minutes to about 3.5 hours of initial drawing of the first sample for performing the nucleic acid analysis.

[0013] The present disclosure further provides a method of screening one or more samples of donor blood for release of the donor blood or material from a donor for clinical use, the method comprising performing a nucleic acid analysis on the one or more samples of donor blood to detect a plurality of pathogens or infectious agents, wherein if a predetermined level of nucleic acid from each of the plurality of pathogens or infectious agents based on the nucleic acid analysis is determined, the method indicates release of the donor blood or donor material for clinical use, and the nucleic acid analysis comprises a nucleic acid amplification reaction having a duration of about 8 minutes to about 20 minutes.

[0014] The present disclosure provides a method for screening one or more samples of donor blood for shipment of the donor blood or material from a donor for clinical use, the method comprising performing a nucleic acid analysis on the one or more samples of donor blood to detect a plurality of pathogens or infectious agents, wherein if a predetermined level of nucleic acid from each of the plurality of pathogens or infectious agents based on the nucleic acid analysis is determined, the method indicates shipment of the donor blood or donor material for clinical use, and wherein each measurement of the predetermined level of nucleic acid from at least one of the plurality of pathogens or infectious agents is completed in about 20 to about 45 minutes from the initial drawing of the one or more samples for performing the nucleic acid analysis.

[0015] The present disclosure provides a method for screening one or more samples of donor blood for release of the donor blood or material from a donor for clinical use, the method comprising performing nucleic acid analysis on the one or more samples of donor blood to detect a plurality of pathogens or infectious agents, wherein if a predetermined level of nucleic acid from each of the plurality of pathogens or infectious agents based on the nucleic acid analysis is determined, release of the donor blood or donor material for clinical use is indicated, and each measurement of the predetermined level of nucleic acid from at least one of the plurality of pathogens or infectious agents has a time-to-result of about 20 minutes to about 45 minutes.

[0016] The present disclosure provides a method of screening one or more samples of donor blood for shipment of the donor blood or material from a donor for clinical use, the method comprising: performing a nucleic acid analysis on the one or more samples of donor blood to detect a plurality of pathogens or infectious agents; and, if a predetermined level of nucleic acid from each of the plurality of pathogens or infectious agents based on the nucleic acid analysis is determined, indicating shipment of the donor blood or donor material for clinical use, and wherein the automated system is configured to screen one or more samples of donor blood for shipment of the donor blood or material from a donor for clinical use. 3 The present invention provides a method that produces at least about 70 results per hour.

[0017] The present disclosure provides a method for screening one or more samples of donor blood for shipment of the donor blood or material from a donor for clinical use, the method comprising: performing a nucleic acid analysis on the one or more samples of donor blood to detect a plurality of pathogens or infectious agents; and indicating shipment of the donor blood or donor material for clinical use if a predetermined level of nucleic acid from each of the plurality of pathogens or infectious agents based on the nucleic acid analysis is determined, the method comprising: 2 The present invention provides a method for generating at least about 140 results per hour per search.

[0018] The present disclosure provides a method of screening one or more samples of donor blood for shipment of the donor blood or material from a donor for clinical use, the method comprising: performing nucleic acid analysis on the one or more samples of donor blood to detect a plurality of pathogens or infectious agents, wherein if a predetermined level of nucleic acid derived from each of the plurality of pathogens or infectious agents based on the nucleic acid analysis is determined, shipment of the donor blood or donor material for clinical use is indicated; and if a presence of nucleic acid derived from at least one of the plurality of pathogens or infectious agents in the pooled sample that exceeds a predetermined level is determined, screening samples of individual donor blood or subpools thereof included in the pooled sample, the method comprising performing nucleic acid analysis on the samples of donor blood to detect the plurality of pathogens or infectious agents, wherein if a predetermined level of nucleic acid derived from each of the plurality of pathogens or infectious agents based on the nucleic acid analysis is determined, shipment of the donor blood or donor material for clinical use is indicated.

[0019] The present disclosure provides a method for screening one or more samples to determine whether donor blood associated with the one or more samples is acceptable for transfusion, the method comprising: performing nucleic acid analysis on the samples, including sample preparation steps and amplification steps; determining results of the nucleic acid analysis; and determining whether the donor blood is acceptable for transfusion based at least in part on the nucleic acid analysis results, wherein shipping of the donor blood or donor material for clinical use occurs in about 15 to about 60 minutes, e.g., about 20 to about 60 minutes, from the initial drawing of one or more samples for performing the nucleic acid analysis.

[0020] The present disclosure also provides a method for screening multiple samples to determine whether donor blood associated with the samples is acceptable for transfusion, the method comprising: performing nucleic acid analysis on the samples, including sample preparation steps and amplification steps; determining results of the nucleic acid analysis; and determining whether the donor blood is acceptable for transfusion based at least in part on the nucleic acid analysis results, wherein the determination based on the nucleic acid analysis is performed within about 15 minutes to about 3.5 hours, e.g., about 20 minutes to about 3.5 hours, of the initial drawing of the first sample for performing the nucleic acid analysis.

[0021] The present disclosure provides a method for screening a plurality of samples to determine whether donor blood associated with the samples is acceptable for transfusion, the method comprising: performing nucleic acid analysis on the samples, including sample preparation steps and amplification steps; determining results of the nucleic acid analysis; and determining whether the donor blood is acceptable for transfusion based at least in part on the nucleic acid analysis results, wherein the nucleic acid analysis comprises a nucleic acid amplification reaction having a duration of about 1 minute to about 20 minutes, e.g., 8 minutes to about 20 minutes.

[0022] The present disclosure further provides a method for screening a plurality of samples to determine whether donor blood associated with the samples is acceptable for transfusion, the method comprising: performing nucleic acid analysis on the samples, including sample preparation steps and amplification steps; determining results of the nucleic acid analysis; and determining whether the donor blood is acceptable for transfusion based at least in part on the nucleic acid analysis results, wherein each determination of a predetermined level of nucleic acid from at least one of a plurality of pathogens or infectious agents is completed in about 15 to about 45 minutes, e.g., about 20 to about 45 minutes, from the initial drawing of the sample to perform the nucleic acid analysis.

[0023] The present disclosure provides a method for screening a plurality of samples to determine whether donor blood associated with the samples is acceptable for transfusion, the method comprising: performing nucleic acid analysis on the samples, including sample preparation and amplification steps; determining results of the nucleic acid analysis; and determining whether the donor blood is acceptable for transfusion based at least in part on the nucleic acid analysis results, wherein each measurement of a predetermined level of nucleic acid from at least one of a plurality of pathogens or infectious agents has a time-to-result of about 15 to about 45 minutes, e.g., about 20 to about 45 minutes.

[0024] The present disclosure provides a method for screening a plurality of samples to determine whether donor blood associated with the samples is acceptable for transfusion, the method comprising: performing nucleic acid analyses on the samples, including sample preparation and amplification steps; determining results of each of the nucleic acid analyses of the samples; and determining whether the donor blood is acceptable for transfusion based at least in part on the nucleic acid analysis results, the method comprising: 3 The present invention provides a method that produces at least about 70 results per hour.

[0025] The present disclosure provides a method for screening a plurality of samples to determine whether donor blood associated with the samples is acceptable for transfusion, the method comprising: performing nucleic acid analyses on the samples, including sample preparation and amplification steps; determining results of each of the nucleic acid analyses of the samples; and determining whether the donor blood is acceptable for transfusion based at least in part on the nucleic acid analysis results, the method comprising: 2 The present invention provides a method for generating at least about 140 results per hour per search.

[0026] In certain embodiments, the nucleic acid analysis comprises a nucleic acid amplification reaction. In certain embodiments, the nucleic acid amplification reaction is an isothermal reaction. In certain embodiments, the nucleic acid analysis comprises optical detection of the presence of nucleic acid from at least one of a plurality of pathogens or infectious agents. In certain embodiments, the nucleic acid analysis comprises digital detection of the presence of nucleic acid from at least one of a plurality of pathogens or infectious agents. In certain embodiments, the nucleic acid analysis comprises simultaneously contacting the sample with a sample lysis buffer and a protease. In certain embodiments, the isothermal reaction is a recombinase polymerase amplification. In certain embodiments, the isothermal reaction is a nicking enzyme amplification reaction. In certain embodiments, the nucleic acid analysis comprises an amplification process, and the amplification process may comprise a nucleic acid amplification reaction. In certain embodiments, the nucleic acid amplification reaction of the amplification process comprises an isothermal reaction. In certain embodiments, the nucleic acid amplification reaction of the amplification process comprises a recombinase polymerase amplification. In certain embodiments, the nucleic acid amplification reaction of the amplification process comprises a nicking enzyme amplification reaction. In certain embodiments, the nucleic acid analysis comprises a detection process. In certain embodiments, the detection process can include optical detection of the presence or absence of nucleic acid from at least one of the multiple pathogens or infectious agents. In certain embodiments, the detection process can include digital detection of the presence or absence of nucleic acid from at least one of the multiple pathogens or infectious agents. In certain embodiments, the nucleic acid analysis includes an amplification and detection process. In certain embodiments, the amplification and detection process can include a nucleic acid amplification reaction and optical detection of the presence or absence of nucleic acid from at least one of the multiple pathogens or infectious agents. In certain embodiments, the amplification and detection process can include a nucleic acid amplification reaction and digital detection of the presence or absence of nucleic acid from at least one of the multiple pathogens or infectious agents. In certain embodiments, the nucleic acid analysis includes a sample preparation process. In certain embodiments, the sample preparation process can include simultaneous contacting of the sample with a sample lysis buffer, optionally with a protease, such as proteinase K.In certain embodiments, the sample preparation process comprises a lysis process, which can include simultaneous contacting of the sample with a sample lysis buffer, optionally with a protease. In certain embodiments, the sample preparation process comprises a lysis process, a pretreatment lysis process, and / or an on-board pooling process, as further described herein.

[0027] In certain embodiments, the plurality of pathogens or infectious agents are selected from the group consisting of SARS-CoV-2 (COVID-19), HIV-1, HIV-2, HBV, HCV, CMV, parvovirus B19, HAV, chlamydia, gonorrhea, WNV, Zika virus, dengue virus, chikungunya virus, influenza, babesia, malaria, Ustu virus, and HEV. Additionally or alternatively, in certain embodiments, the plurality of pathogens or infectious agents may include one or more emerging pathogens, viruses, and / or agents.

[0028] In certain embodiments, the multiple pathogens or infectious agents are HIV-1, HIV-2, HCV, and HBV. In certain embodiments, the multiple pathogens or infectious agents are HIV-1 and HBV. In certain embodiments, the multiple pathogens or infectious agents are HIV-2 and HCV. In certain embodiments, the multiple pathogens or infectious agents are HIV-1, HIV-2, HCV, HBV, and WNV. In certain embodiments, the multiple pathogens or infectious agents are Zika virus and WNV. In certain embodiments, the multiple pathogens or infectious agents are Chikungunya virus and Dengue virus. In certain embodiments, the multiple pathogens or infectious agents are Zika virus, WNV, Chikungunya virus, and Dengue virus. In certain embodiments, the multiple pathogens or infectious agents are Babesia and Malaria. In certain embodiments, the multiple pathogens or infectious agents are Parvovirus B19 and HAV. In certain embodiments, the plurality of pathogens or infectious agents are HIV-1, HIV-2, HCV, HBV, WNV, and Zika virus. In certain embodiments, the plurality of pathogens or infectious agents are HIV-1, HIV-2, HCV, HBV, WNV, Zika virus, and Chikungunya virus. In certain embodiments, the plurality of pathogens or infectious agents are HIV-1, HIV-2, HCV, HBV, WNV, Zika virus, Chikungunya virus, and Dengue virus. In certain embodiments, the plurality of pathogens or infectious agents are HIV-1, HIV-2, HCV, HBV, WNV, Zika virus, Chikungunya virus, Dengue virus, and Babesia. In certain embodiments, the plurality of pathogens or infectious agents are HIV-1, HIV-2, HCV, HBV, WNV, Zika virus, Chikungunya virus, Dengue virus, Babesia, and malaria. In certain embodiments, the multiple pathogens or infectious agents are HIV-1, HIV-2, HCV, HBV, WNV, Zika virus, Chikungunya virus, Dengue virus, Babesia, Malaria, and Parvovirus B19.In certain embodiments, the plurality of pathogens or infectious agents are HIV-1, HIV-2, HCV, HBV, WNV, Zika virus, chikungunya virus, dengue virus, babesia, malaria, parvovirus B19, and HAV. In certain embodiments, the plurality of pathogens or infectious agents are HIV-1, HIV-2, HCV, HBV, WNV, Zika virus, chikungunya virus, dengue virus, babesia, malaria, parvovirus B19, HAV, and HEV. In certain embodiments, the plurality of pathogens or infectious agents are HIV-1, HIV-2, HCV, HBV, and parvovirus B19. In certain embodiments, the plurality of pathogens or infectious agents are HIV-1, HIV-2, HCV, HBV, parvovirus B19, and HAV. In certain embodiments, the plurality of pathogens or infectious agents are HIV-1, HIV-2, HCV, HBV, parvovirus B19, and HAV. In certain embodiments, the plurality of pathogens or infectious agents are HIV-1, HIV-2, HCV, HBV, and HAV. In certain embodiments, the plurality of pathogens or infectious agents are HIV-1, HIV-2, HCV, HBV, and HEV. In certain embodiments, the plurality of pathogens or infectious agents are HIV-1, HIV-2, HCV, HBV, and Zika virus. In certain embodiments, the plurality of pathogens or infectious agents are HIV-1, HIV-2, HCV, HBV, Zika virus, and dengue virus. In certain embodiments, the plurality of pathogens or infectious agents are HIV-1, HIV-2, HCV, HBV, Zika virus, dengue virus, and chikungunya virus. In certain embodiments, the plurality of pathogens or infectious agents are HIV-1, HIV-2, HCV, HBV, Zika virus, dengue virus, and WNV. In certain embodiments, the multiple pathogens or infectious agents are HIV-1, HIV-2, HCV, HBV, Zika virus, WNV, and Chikungunya virus. In certain embodiments, the multiple pathogens or infectious agents are HIV-1, HIV-2, HCV, HBV, Zika virus, WNV, Dengue virus, and Chikungunya virus.In certain embodiments, the plurality of pathogens or infectious agents are HIV-1, HIV-2, HCV, HBV, and malaria. In certain embodiments, the plurality of pathogens or infectious agents are HIV-1, HIV-2, HCV, HBV, malaria, and Babesia. In certain embodiments, the plurality of pathogens or infectious agents are HIV-1, HIV-2, HCV, HBV, and dengue virus. In certain embodiments, the plurality of pathogens or infectious agents are HIV-1, HIV-2, HCV, HBV, dengue virus, and chikungunya virus. In certain embodiments, the plurality of pathogens or infectious agents are HIV-1, HIV-2, HCV, HBV, dengue virus, WNV, and chikungunya virus. In certain embodiments, the plurality of pathogens or infectious agents are HIV-1, HIV-2, HCV, HBV, dengue virus, WNV, and chikungunya virus. In certain embodiments, the plurality of pathogens or infectious agents are HIV-1, HIV-2, HCV, HBV, chikungunya virus, and Zika virus. In certain embodiments, the plurality of pathogens or infectious agents are HIV-1, HIV-2, HCV, HBV, chikungunya virus, Zika virus, and WNV. In certain embodiments, the plurality of pathogens or infectious agents are HIV-1, HIV-2, HCV, HBV, WNV, and dengue virus. In certain embodiments, the plurality of pathogens or infectious agents are HIV-1, HIV-2, HCV, HBV, chikungunya virus, Zika virus, and WNV.

[0029] In certain embodiments, the plurality of pathogens or infectious agents are HIV-1, HIV-2, HCV, and HBV; and the nucleic acid analysis comprises multiplex analysis of HIV-1 and HIV-2. In certain embodiments, the plurality of pathogens or infectious agents are HIV-1, HIV-2, HCV, and HBV; and the nucleic acid analysis comprises multiplex analysis of HIV-1, HIV-2, and HCV. In certain embodiments, the plurality of pathogens or infectious agents are HIV-1, HIV-2, HCV, and HBV; and the nucleic acid analysis comprises multiplex analysis of HIV-1, HIV-2, and HBV. In certain embodiments, the plurality of pathogens or infectious agents are HIV-1, HIV-2, HCV, and HBV; and the nucleic acid analysis comprises multiplex analysis of HCV and HBV. In certain embodiments, the plurality of pathogens or infectious agents are HIV-1, HIV-2, HCV, and HBV; the nucleic acid analysis comprises multiplex analysis of HIV-1 and HIV-2; and the nucleic acid analysis comprises multiplex analysis of HCV and HBV. In certain embodiments, the plurality of pathogens or infectious agents are HIV-1, HIV-2, HCV, and HBV; and the nucleic acid analysis comprises multiplex analysis of HIV-1, HIV-2, HCV, and HBV.

[0030] In certain embodiments, the plurality of pathogens or infectious agents comprises Zika virus, WNV, chikungunya virus, and dengue virus; and the nucleic acid analysis comprises multiplex analysis of Zika virus and WNV. In certain embodiments, the plurality of pathogens or infectious agents comprises Zika virus, WNV, chikungunya virus, and dengue virus; and the nucleic acid analysis comprises multiplex analysis of chikungunya virus and dengue virus. In certain embodiments, the plurality of pathogens or infectious agents comprises Zika virus, WNV, chikungunya virus, and dengue virus; and the nucleic acid analysis comprises multiplex analysis of Zika virus and WNV; and the nucleic acid analysis comprises multiplex analysis of chikungunya virus and dengue virus. In certain embodiments, the plurality of pathogens or infectious agents comprises Zika virus, WNV, chikungunya virus, and dengue virus; and the nucleic acid analysis comprises multiplex analysis of chikungunya virus and WNV; and the nucleic acid analysis comprises multiplex analysis of Zika virus and dengue virus. In certain embodiments, the plurality of pathogens or infectious agents comprises Zika virus, WNV, Chikungunya virus, and Dengue virus; the nucleic acid analysis comprises multiplex analysis of Zika virus and Dengue virus; the nucleic acid analysis comprises multiplex analysis of Chikungunya virus and WNV. In certain embodiments, the plurality of pathogens or infectious agents comprises Babesia and Malaria; the nucleic acid analysis comprises multiplex analysis of Babesia and Malaria. In certain embodiments, the plurality of pathogens or infectious agents comprises Parvovirus B19 and HAV; the nucleic acid analysis comprises multiplex analysis of Parvovirus B19 and HAV.

[0031] In certain embodiments, nucleic acid analysis involves contacting a sample with CuTi-coated microparticles. In certain embodiments, nucleic acid analysis involves contacting a sample with a plurality of microparticles and translating the microparticles across a surface via magnetic force. In certain embodiments, nucleic acid analysis involves purifying nucleic acids from a sample of donor blood; dividing the purified nucleic acids into a plurality of fractions; and retaining at least one fraction for further screening. In certain embodiments, nucleic acid analysis involves a sample preparation process, which can include contacting the sample with CuTi-coated microparticles. By way of example and not limitation, the sample preparation process can include a lysis process, which can include contacting the sample with CuTi-coated microparticles. In certain embodiments, the sample preparation process can include a washing process. For example, and not by way of limitation, the washing process can include translating the microparticles across a surface via magnetic force. In certain embodiments, the sample preparation process can be performed in a sample preparation area. In certain embodiments, the sample preparation area can include a sample transporter, e.g., a sample preparation carousel, and a washing and elution system. In certain embodiments, the sample preparation area can include a particle transfer mechanism that can transfer CuTi-coated microparticles from a sample transport, such as a sample preparation carousel, to a wash and elution system. For example, the particle transfer mechanism can be a magnetic force that transfers CuTi-coated microparticles, such as via a magnetic tip.

[0032] In certain embodiments, the plurality of pathogens or infectious agents and predetermined levels include SARS-CoV-2 (COVID-19) at a predetermined level of at least 1-50 copies / mL; HIV-1 at a predetermined level of at least 1-50 copies / mL; HIV-2 at a predetermined level of at least 1-20 IU / mL; HBV at a predetermined level of at least 1-10 IU / mL; HCV at a predetermined level of at least 1-50 IU / mL; CMV at a predetermined level of at least 10-50 IU / mL; Parvovirus B19 at a predetermined level of at least 1-40 IU / mL; HAV at a predetermined level of at least 1-10 IU / mL; chlamydia at a predetermined level; gonorrhea at a predetermined level of at least 100-500 copies / mL; WNV at a predetermined level of at least 1-50 copies / mL; Zika virus at a predetermined level of at least 1-50 copies / mL; dengue virus at a predetermined level of at least 1-50 copies / mL; chikungunya virus at a predetermined level of at least 1-50 copies / mL; influenza at a predetermined level of at least 10-500 copies / mL; Babesia at a predetermined level of at least 1-20 copies / mL; malaria at a predetermined level of at least 1-50 copies / mL; and HEV at a predetermined level of at least 1-20 IU / mL.

[0033] In certain embodiments, the plurality of pathogens or infectious agents are HIV-1, HIV-2, HCV, and HBV; the predetermined levels are at least 1-50 copies / mL of HIV-1; at least 1-20 IU / mL of HIV-2; at least 1-50 IU / mL of HCV; and at least 1-10 IU / mL of HBV. In certain embodiments, the plurality of pathogens or infectious agents are HIV-1, HIV-2, HCV, HBV, and WNV; the predetermined levels are at least 1-50 copies / mL of HIV-1; at least 1-20 IU / mL of HIV-2; at least 1-50 IU / mL of HCV; at least 1-10 IU / mL of HBV; and at least 1-50 copies / mL of WNV. In certain embodiments, the plurality of pathogens or infectious agents are Zika virus and WNV; the predetermined levels are at least 1-50 copies / mL of Zika virus and at least 1-50 copies / mL of WNV. In certain embodiments, the plurality of pathogens or infectious agents are chikungunya virus and dengue virus; the predetermined levels are at least 1-50 copies / mL of chikungunya virus; and at least 1-50 copies / mL of dengue virus. In certain embodiments, the plurality of pathogens or infectious agents are Zika virus, WNV, chikungunya virus, and dengue virus; the predetermined levels are at least 1-50 copies / mL of Zika virus; at least 1-50 copies / mL of WNV; at least 1-50 copies / mL of chikungunya virus; and at least 1-50 copies / mL of dengue virus.

[0034] In certain embodiments, the plurality of pathogens or infectious agents are Babesia and Malaria; the predetermined levels are at least 1-20 copies / mL of Babesia; and at least 1-50 copies / mL of Malaria. In certain embodiments, the plurality of pathogens or infectious agents are Parvovirus B19 and HAV; the predetermined levels are at least 1-40 IU / mL of Parvovirus B19; and at least 1-10 IU / mL of HAV. In certain embodiments, the plurality of pathogens or infectious agents are HIV-1, HIV-2, HCV, HBV, WNV, and Zika virus; the predetermined levels are at least 1-50 copies / mL of HIV-1; at least 1-20 IU / mL of HIV-2; at least 1-50 IU / mL of HCV; at least 1-10 IU / mL of HBV; at least 1-50 copies / mL of WNV; and at least 1-50 copies / mL of Zika virus. In certain embodiments, the multiple pathogens or infectious agents are HIV-1, HIV-2, HCV, HBV, WNV, Zika virus, and Chikungunya virus, and the predetermined levels are at least 1-50 copies / mL of HIV-1; at least 1-20 IU / mL of HIV-2; at least 1-50 IU / mL of HCV; at least 1-10 IU / mL of HBV; at least 1-50 copies / mL of WNV; at least 1-50 copies / mL of Zika virus; and at least 1-50 copies / mL of Chikungunya virus. In certain embodiments, the plurality of pathogens or infectious agents are HIV-1, HIV-2, HCV, HBV, WNV, Zika virus, Chikungunya virus, and Dengue virus; and the predetermined levels are at least 1-50 copies / mL of HIV-1; at least 1-20 IU / mL of HIV-2; at least 1-50 IU / mL of HCV; at least 1-10 IU / mL of HBV; at least 1-50 copies / mL of WNV; at least 1-50 copies / mL of Zika virus; at least 1-50 copies / mL of Chikungunya virus; and at least 1-50 copies / mL of Dengue virus.

[0035] In certain embodiments, the plurality of pathogens or infectious agents are HIV-1, HIV-2, HCV, HBV, WNV, Zika virus, Chikungunya virus, Dengue virus, and Babesia; and the predetermined levels are at least 1-50 copies / mL of HIV-1; at least 1-20 IU / mL of HIV-2; at least 1-50 IU / mL of HCV; at least 1-10 IU / mL of HBV; at least 1-50 copies / mL of WNV; at least 1-50 copies / mL of Zika virus; at least 1-50 copies / mL of Chikungunya virus; at least 1-50 copies / mL of Dengue virus; and at least 1-20 copies / mL of Babesia. In certain embodiments, the plurality of pathogens or infectious agents are HIV-1, HIV-2, HCV, HBV, WNV, Zika virus, Chikungunya virus, Dengue virus, Babesia, and Malaria; and the predetermined levels are at least 1-50 copies / mL of HIV-1; at least 1-20 IU / mL of HIV-2; at least 1-50 IU / mL of HCV; at least 1-10 IU / mL of HBV; at least 1-50 copies / mL of WNV; at least 1-50 copies / mL of Zika virus; at least 1-50 copies / mL of Chikungunya virus; at least 1-50 copies / mL of Dengue virus; at least 1-20 copies / mL of Babesia; and at least 1-50 copies / mL of Malaria. In certain embodiments, the plurality of pathogens or infectious agents are HIV-1, HIV-2, HCV, HBV, WNV, Zika virus, Chikungunya virus, Dengue virus, Babesia, malaria, and Parvovirus B19; and the predetermined levels are at least 1-50 copies / mL of HIV-1; at least 1-20 IU / mL of HIV-2; at least 1-50 IU / mL of HCV; at least 1-10 IU / mL of HBV; at least 1-50 copies / mL of WNV; at least 1-50 copies / mL of Zika virus; at least 1-50 copies / mL of Chikungunya virus; at least 1-50 copies / mL of Dengue virus; at least 1-20 copies / mL of Babesia; at least 1-50 copies / mL of Malaria; and at least 1-40 IU / mL of Parvovirus B19.In certain embodiments, the plurality of pathogens or infectious agents are HIV-1, HIV-2, HCV, HBV, WNV, Zika virus, Chikungunya virus, Dengue virus, Babesia, malaria, Parvovirus B19, and HAV; and the predetermined levels are at least 1-50 copies / mL of HIV-1; at least 1-20 IU / mL of HIV-2; at least 1-50 IU / mL of HCV; at least 1-10 IU / mL of HBV; at least 1-50 copies / mL of WNV; at least 1-50 copies / mL of Zika virus; at least 1-50 copies / mL of Chikungunya virus; at least 1-50 copies / mL of Dengue virus; at least 1-20 copies / mL of Babesia; at least 1-50 copies / mL of Malaria; at least 1-40 IU / mL of Parvovirus B19; and at least 1-10 IU / mL of HAV. In certain embodiments, the plurality of pathogens or infectious agents are HIV-1, HIV-2, HCV, HBV, WNV, Zika virus, Chikungunya virus, Dengue virus, Babesia, malaria, Parvovirus B19, HAV, and HEV; and the predetermined levels are at least 1-50 copies / mL of HIV-1; at least 1-20 IU / mL of HIV-2; at least 1-50 IU / mL of HCV; at least 1-10 IU / mL of HBV; and at least and at least 1-50 copies / mL of WNV; at least 1-50 copies / mL of Zika virus; at least 1-50 copies / mL of Chikungunya virus; at least 1-50 copies / mL of Dengue virus; at least 1-20 copies / mL of Babesia; at least 1-50 copies / mL of Malaria; and at least 1-40 IU / mL of Parvovirus B19; at least 1-10 IU / mL of HAV; and at least 1-20 IU / mL of HEV.

[0036] In certain embodiments, the plurality of pathogens or infectious agents are HIV-1, HIV-2, HCV, HBV, and parvovirus B19; and the predetermined levels are at least 1-50 copies / mL of HIV-1, at least 1-20 IU / mL of HIV-2, at least 1-50 IU / mL of HCV, at least 1-10 IU / mL of HBV, and at least 1-40 IU / mL of parvovirus B19. In certain embodiments, the plurality of pathogens or infectious agents are HIV-1, HIV-2, HCV, HBV, parvovirus B19, and HAV; and the predetermined levels are at least 1-50 copies / mL of HIV-1, at least 1-20 IU / mL of HIV-2, at least 1-50 IU / mL of HCV, at least 1-10 IU / mL of HBV, at least 1-40 IU / mL of parvovirus B19, and at least 1-10 IU / mL of HAV. In certain embodiments, the plurality of pathogens or infectious agents are HIV-1, HIV-2, HCV, HBV, and Babesia; the predetermined levels are at least 1-50 copies / mL of HIV-1, at least 1-20 IU / mL of HIV-2, at least 1-50 IU / mL of HCV, at least 1-10 IU / mL of HBV, and at least 1-20 copies / mL of Babesia. In certain embodiments, the plurality of pathogens or infectious agents are HIV-1, HIV-2, HCV, HBV, and HAV; the predetermined levels are at least 1-50 copies / mL of HIV-1, at least 1-20 IU / mL of HIV-2, at least 1-50 IU / mL of HCV, at least 1-10 IU / mL of HBV, and at least 1-10 IU / mL of HAV. In certain embodiments, the multiple pathogens or infectious agents are HIV-1, HIV-2, HCV, HBV, and HEV; and the predetermined levels are at least 1-50 copies / mL of HIV-1; at least 1-20 IU / mL of HIV-2; at least 1-50 IU / mL of HCV; at least 1-10 IU / mL of HBV; and at least 1-20 IU / mL of HEV.In certain embodiments, the multiple pathogens or infectious agents are HIV-1, HIV-2, HCV, HBV, and Zika virus; and the predetermined levels are at least 1-50 copies / mL of HIV-1; at least 1-20 IU / mL of HIV-2; at least 1-50 IU / mL of HCV; at least 1-10 IU / mL of HBV; and at least 1-50 copies / mL of Zika virus.

[0037] In certain embodiments, the multiple pathogens or infectious agents are HIV-1, HIV-2, HCV, HBV, Zika virus, and Dengue virus; and the predetermined levels are at least 1-50 copies / mL of HIV-1; at least 1-20 IU / mL of HIV-2; at least 1-50 IU / mL of HCV; at least 1-10 IU / mL of HBV; at least 1-50 copies / mL of Zika virus; and at least 1-50 copies / mL of Dengue virus. In certain embodiments, the plurality of pathogens or infectious agents are HIV-1, HIV-2, HCV, HBV, Zika virus, dengue virus, and chikungunya virus; and the predetermined levels are at least 1-50 copies / mL of HIV-1; at least 1-20 IU / mL of HIV-2; at least 1-50 IU / mL of HCV; at least 1-10 IU / mL of HBV; at least 1-50 copies / mL of Zika virus; at least 1-50 copies / mL of dengue virus; and at least 1-50 copies / mL of chikungunya virus. In certain embodiments, the plurality of pathogens or infectious agents are HIV-1, HIV-2, HCV, HBV, Zika virus, dengue virus, and WNV; and the predetermined levels are at least 1-50 copies / mL of HIV-1; at least 1-20 IU / mL of HIV-2; at least 1-50 IU / mL of HCV; at least 1-10 IU / mL of HBV; at least 1-50 copies / mL of Zika virus; at least 1-50 copies / mL of dengue virus; and at least 1-50 copies / mL of WNV. In certain embodiments, the plurality of pathogens or infectious agents are HIV-1, HIV-2, HCV, HBV, Zika virus, WNV, and Chikungunya virus; and the predetermined levels are at least 1-50 copies / mL of HIV-1; at least 1-20 IU / mL of HIV-2; at least 1-50 IU / mL of HCV; at least 1-10 IU / mL of HBV; at least 1-50 copies / mL of Zika virus; at least 1-50 copies / mL of WNV; and at least 1-50 copies / mL of Chikungunya virus.In certain embodiments, the plurality of pathogens or infectious agents are HIV-1, HIV-2, HCV, HBV, Zika virus, WNV, dengue virus, and chikungunya virus; and the predetermined levels are at least 1-50 copies / mL of HIV-1; at least 1-20 IU / mL of HIV-2; at least 1-50 IU / mL of HCV; at least 1-10 IU / mL of HBV; at least 1-50 copies / mL of Zika virus; at least 1-50 copies / mL of WNV; at least 1-50 copies / mL of dengue virus; and at least 1-50 copies / mL of Chikungunya virus.

[0038] In certain embodiments, the plurality of pathogens or infectious agents are HIV-1, HIV-2, HCV, HBV, and malaria; the predetermined levels are at least 1-50 copies / mL of HIV-1, at least 1-20 IU / mL of HIV-2, at least 1-50 IU / mL of HCV, at least 1-10 IU / mL of HBV, and at least 1-50 copies / mL of malaria. In certain embodiments, the plurality of pathogens or infectious agents are HIV-1, HIV-2, HCV, HBV, malaria, and babesia; the predetermined levels are at least 1-50 copies / mL of HIV-1, at least 1-20 IU / mL of HIV-2, at least 1-50 IU / mL of HCV, at least 1-10 IU / mL of HBV, at least 1-50 copies / mL of malaria, and at least 1-20 copies / mL of babesia. In certain embodiments, the plurality of pathogens or infectious agents are HIV-1, HIV-2, HCV, HBV, and dengue virus; the predetermined levels are at least 1-50 copies / mL of HIV-1, at least 1-20 IU / mL of HIV-2, at least 1-50 IU / mL of HCV, at least 1-10 IU / mL of HBV, and at least 1-50 copies / mL of dengue virus. In certain embodiments, the plurality of pathogens or infectious agents are HIV-1, HIV-2, HCV, HBV, dengue virus, and chikungunya virus; the predetermined levels are at least 1-50 copies / mL of HIV-1, at least 1-20 IU / mL of HIV-2, at least 1-50 IU / mL of HCV, at least 1-10 IU / mL of HBV, at least 1-50 copies / mL of dengue virus, and at least 1-50 copies / mL of chikungunya virus.In certain embodiments, the multiple pathogens or infectious agents are HIV-1, HIV-2, HCV, HBV, dengue virus, WNV, and chikungunya virus; and the predetermined levels are at least 1-50 copies / mL of HIV-1; at least 1-20 IU / mL of HIV-2; at least 1-50 IU / mL of HCV; at least 1-10 IU / mL of HBV; at least 1-50 copies / mL of dengue virus; at least 1-50 copies / mL of WNV; and at least 1-50 copies / mL of chikungunya virus. In certain embodiments, the multiple pathogens or infectious agents are HIV-1, HIV-2, HCV, HBV, and Chikungunya virus; and the predetermined levels are at least 1-50 copies / mL of HIV-1; at least 1-20 IU / mL of HIV-2; at least 1-50 IU / mL of HCV; at least 1-10 IU / mL of HBV; and at least 1-50 copies / mL of Chikungunya virus.

[0039] In certain embodiments, the plurality of pathogens or infectious agents are HIV-1, HIV-2, HCV, HBV, Chikungunya virus, and Zika virus; and the predetermined levels are at least 1-50 copies / mL of HIV-1; at least 1-20 IU / mL of HIV-2; at least 1-50 IU / mL of HCV; at least 1-10 IU / mL of HBV; at least 1-50 copies / mL of Chikungunya virus; and at least 1-50 copies / mL of Zika virus. In certain embodiments, the plurality of pathogens or infectious agents are HIV-1, HIV-2, HCV, HBV, Chikungunya virus, Zika virus, and WNV; and the predetermined levels are at least 1-50 copies / mL of HIV-1; at least 1-20 IU / mL of HIV-2; at least 1-50 IU / mL of HCV; at least 1-10 IU / mL of HBV; at least 1-50 copies / mL of Chikungunya virus; at least 1-50 copies / mL of Zika virus; and at least 1-50 copies / mL of WNV. In certain embodiments, the plurality of pathogens or infectious agents are HIV-1, HIV-2, HCV, HBV, WNV, and dengue virus; the predetermined levels are at least 1-50 copies / mL of HIV-1, at least 1-20 IU / mL of HIV-2, at least 1-50 IU / mL of HCV, at least 1-10 IU / mL of HBV, at least 1-50 copies / mL of WNV, and at least 1-50 copies / mL of dengue virus. In certain embodiments, the plurality of pathogens or infectious agents are HIV-1, HIV-2, HCV, and HBV; the nucleic acid analysis includes multiplex analysis of HIV-1 and HIV-2; the predetermined levels are at least 1-50 copies / mL of HIV-1, at least 1-20 IU / mL of HIV-2, at least 1-50 IU / mL of HCV, and at least 1-10 IU / mL of HBV.In certain embodiments, the plurality of pathogens or infectious agents are HIV-1, HIV-2, HCV, and HBV; the nucleic acid analysis comprises multiplex analysis of HIV-1, HIV-2, and HCV; and the predetermined levels are at least 1-50 copies / mL of HIV-1, at least 1-20 IU / mL of HIV-2, at least 1-50 IU / mL of HCV, and at least 1-10 IU / mL of HBV. In certain embodiments, the plurality of pathogens or infectious agents are HIV-1, HIV-2, HCV, and HBV; the nucleic acid analysis comprises multiplex analysis of HIV-1, HIV-2, and HBV; and the predetermined levels are at least 1-50 copies / mL of HIV-1, at least 1-20 IU / mL of HIV-2, at least 1-50 IU / mL of HCV, and at least 1-10 IU / mL of HBV.

[0040] In certain embodiments, the plurality of pathogens or infectious agents are HIV-1, HIV-2, HCV, and HBV; the nucleic acid analysis comprises multiplex analysis of HCV and HBV; and the predetermined levels are at least 1-50 copies / mL of HIV-1, at least 1-20 IU / mL of HIV-2, at least 1-50 IU / mL of HCV, and at least 1-10 IU / mL of HBV. In certain embodiments, the plurality of pathogens or infectious agents are HIV-1, HIV-2, HCV, and HBV; the nucleic acid analysis comprises multiplex analysis of HIV-1 and HIV-2; the nucleic acid analysis comprises multiplex analysis of HCV and HBV; and the predetermined levels are at least 1-50 copies / mL of HIV-1, at least 1-20 IU / mL of HIV-2, at least 1-50 IU / mL of HCV, and at least 1-10 IU / mL of HBV. In certain embodiments, the plurality of pathogens or infectious agents are HIV-1, HIV-2, HCV, and HBV; the nucleic acid analysis comprises multiplex analysis of HIV-1, HIV-2, HCV, and HBV; and the predetermined levels are at least 1-50 copies / mL of HIV-1, at least 1-20 IU / mL of HIV-2, at least 1-50 IU / mL of HCV, and at least 1-10 IU / mL of HBV. In certain embodiments, the plurality of pathogens or infectious agents are Zika virus, WNV, Chikungunya virus, and Dengue virus; the nucleic acid analysis comprises multiplex analysis of Zika virus and WNV; and the predetermined levels are at least 1-50 copies / mL of Zika virus, at least 1-50 copies / mL of WNV, at least 1-50 copies / mL of Chikungunya virus, and at least 1-50 copies / mL of Dengue virus. In certain embodiments, the multiple pathogens or infectious agents are Zika virus, WNV, Chikungunya virus, and Dengue virus; the nucleic acid analysis includes multiplex analysis of Chikungunya virus and Dengue virus; and the predetermined levels are at least 1-50 copies / mL of Zika virus; at least 1-50 copies / mL of WNV; at least 1-50 copies / mL of Chikungunya virus; and at least 1-50 copies / mL of Dengue virus.In certain embodiments, the multiple pathogens or infectious agents are Zika virus, WNV, Chikungunya virus, and Dengue virus; the nucleic acid analysis includes multiplex analysis of Zika virus and WNV; the nucleic acid analysis includes multiplex analysis of Chikungunya virus and Dengue virus; and the predetermined levels are at least 1-50 copies / mL of Zika virus; at least 1-50 copies / mL of WNV; at least 1-50 copies / mL of Chikungunya virus; and at least 1-50 copies / mL of Dengue virus.

[0041] In certain embodiments, the multiple pathogens or infectious agents are Zika virus, WNV, Chikungunya virus, and Dengue virus; the nucleic acid analysis includes multiplex analysis of Chikungunya virus and WNV; the nucleic acid analysis includes multiplex analysis of Zika virus and Dengue virus; and the predetermined levels are at least 1-50 copies / mL of Zika virus; at least 1-50 copies / mL of WNV; at least 1-50 copies / mL of Chikungunya virus; and at least 1-50 copies / mL of Dengue virus. In certain embodiments, the plurality of pathogens or infectious agents are Zika virus, WNV, chikungunya virus, and dengue virus; the nucleic acid analysis comprises multiplex analysis of Zika virus and dengue virus; the nucleic acid analysis comprises multiplex analysis of chikungunya virus and WNV; and the predetermined levels are at least 1-50 copies / mL of Zika virus; at least 1-50 copies / mL of WNV; at least 1-50 copies / mL of chikungunya virus; and at least 1-50 copies / mL of dengue virus. In certain embodiments, the plurality of pathogens or infectious agents comprise Babesia and malaria; the nucleic acid analysis comprises multiplex analysis of Babesia and malaria; and the predetermined levels are at least 1-50 copies / mL of malaria; and at least 1-20 copies / mL of Babesia. In certain embodiments, the multiple pathogens or infectious agents include parvovirus B19 and HAV; the nucleic acid analysis includes multiplex analysis of parvovirus B19 and HAV; and the predetermined levels are at least 1-40 IU / mL of parvovirus B19; and at least 1-10 IU / mL of HAV.

[0042] In certain embodiments, the sample of donor blood is human donor blood. In certain embodiments, the sample of donor blood is whole blood. In certain embodiments, the sample of donor blood is lysed whole blood. In certain embodiments, the sample of donor blood is serum. In certain embodiments, the sample of donor blood is plasma.

[0043] In certain embodiments, the donor blood sample is pooled. In certain embodiments, the pooled donor blood sample includes blood from two donors. In certain embodiments, the pooled donor blood sample includes blood from three donors. In certain embodiments, the pooled donor blood sample includes blood from four donors. In certain embodiments, the pooled donor blood sample includes blood from five donors. In certain embodiments, the pooled donor blood sample includes blood from six donors. In certain embodiments, the pooled donor blood sample includes blood from eight donors. In certain embodiments, the pooled donor blood sample includes blood from ten donors. In certain embodiments, the pooled donor blood sample includes blood from twelve donors. In certain embodiments, the pooled donor blood sample includes blood from eighteen donors. In certain embodiments, the pooled donor blood sample includes blood from twenty-four donors. In certain embodiments, the pooled donor blood sample includes blood from forty-eight donors. In one particular embodiment, the pooled sample of donor blood comprises blood from 96 donors.

[0044] In certain embodiments, the shipment of donor blood is for transfusion. In certain embodiments, the shipment of donor blood is for use in medicine. In certain embodiments, the shipment of donor blood is for use in therapeutic treatment. In certain embodiments, the sample of donor blood is for use as a blood donation. In certain embodiments, the clinical use is blood transfusion. In certain embodiments, the clinical use is use in medicine. In certain embodiments, the clinical use is use in therapeutic treatment. In certain embodiments, the clinical use is use in disease diagnosis or quality assurance / laboratory diagnostics.

[0045] The present disclosure provides an automated system for screening one or more samples of donor blood for shipment for clinical use. In certain embodiments, the automated system includes a sample analysis station including a sample loading area, a sample preparation area, a nucleic acid amplification area, and a nucleic acid detection area; a processor; and memory containing instructions that, when executed by the processor, cause the system to perform nucleic acid analysis on one or more samples of donor blood to detect one or more of a plurality of pathogens or infectious agents. If a predetermined level of nucleic acid derived from each of the plurality of pathogens or infectious agents based on the nucleic acid analysis is determined, the system indicates shipment of the donor blood or donor material for clinical use, and the shipment of the donor blood or donor material for clinical use occurs about 15 to about 60 minutes, e.g., about 20 to about 60 minutes, from the initial drawing of one or more samples for performing the nucleic acid analysis. In certain embodiments, the nucleic acid amplification area and the nucleic acid detection area can be a single area, e.g., an amplification and detection system.

[0046] The present disclosure also provides an automated system for screening one or more samples of donor blood for shipment of the donor blood or donor material for clinical use, the automated system comprising: a sample analysis station including a sample loading area, a sample preparation area, a nucleic acid amplification area, and a nucleic acid detection area; a processor; and memory, the memory including instructions, when executed by the processor, for performing nucleic acid analysis on the one or more samples of donor blood to detect one or more of a plurality of pathogens or infectious agents; if a predetermined level of nucleic acid from each of the plurality of pathogens or infectious agents based on the nucleic acid analysis is determined, the system indicates shipment of the donor blood or donor material for clinical use; the system is configured to screen multiple samples of donor blood for shipment of the donor blood or donor material for clinical use, and makes a determination based on the nucleic acid analysis within about 15 minutes to about 3.5 hours, for example, within about 20 minutes to about 3.5 hours, of the initial drawing of a first sample for performing the nucleic acid analysis.

[0047] The present disclosure further provides an automated system for screening one or more samples of donor blood for release to clinical use, the automated system comprising: a sample analysis station including a sample loading area, a sample preparation area, a nucleic acid amplification area, and a nucleic acid detection area; a processor; and a memory, the memory including instructions, when executed by the processor, for performing nucleic acid analysis on the one or more samples of donor blood to detect one or more of a plurality of pathogens or infectious agents; if a predetermined level of nucleic acid derived from each of the plurality of pathogens or infectious agents based on the nucleic acid analysis is determined, the system indicates release to clinical use of the donor blood or donor material, and the nucleic acid analysis includes a nucleic acid amplification reaction having a duration of about 1 minute to about 20 minutes, for example, about 8 minutes to about 20 minutes.

[0048] The present disclosure provides an automated system for screening one or more samples of donor blood for shipment for clinical use, the automated system comprising: a sample analysis station including a sample loading area, a sample preparation area, a nucleic acid amplification area, and a nucleic acid detection area; a processor; and a memory, the memory including instructions, when executed by the processor, for performing nucleic acid analysis on the one or more samples of donor blood to detect one or more of a plurality of pathogens or infectious agents; if a predetermined level of nucleic acid from each of the plurality of pathogens or infectious agents based on the nucleic acid analysis is determined, the system indicates shipment of the donor blood or donor material for clinical use, wherein each measurement of the predetermined level of nucleic acid from at least one of the plurality of pathogens or infectious agents is completed in about 30 to about 45 minutes from the initial drawing of the sample to perform the nucleic acid analysis.

[0049] The present disclosure provides an automated system for screening one or more samples of donor blood for shipment for clinical use, the automated system comprising: a sample analysis station including a sample loading area, a sample preparation area, a nucleic acid amplification area, and a nucleic acid detection area; a processor; and a memory, the memory including instructions, when executed by the processor, for performing nucleic acid analysis on the one or more samples of donor blood to detect one or more of a plurality of pathogens or infectious agents; if a predetermined level of nucleic acid from each of the plurality of pathogens or infectious agents based on the nucleic acid analysis is determined, the system indicates shipment of the donor blood or donor material for clinical use, wherein each measurement of the predetermined level of nucleic acid from at least one of the plurality of pathogens or infectious agents has a time to result of about 30 minutes to about 45 minutes.

[0050] The present disclosure provides an automated system for screening samples of donor blood for shipment of the donor blood or material from a donor for clinical use, the system comprising: a sample analysis station including a sample loading area, a sample preparation area, a nucleic acid amplification area, and a nucleic acid detection area; a processor; and a memory, the memory including instructions, when executed by the processor, for performing nucleic acid analysis on one or more samples of the donor blood to detect one or more of a plurality of pathogens or infectious agents; and if the nucleic acid analysis results in a determination of a predetermined level of nucleic acid from each of the plurality of pathogens or infectious agents, the system indicates shipment of the donor blood or donor material for clinical use, and the automated system occupies a volume of 1 m 3 To provide an automated system capable of producing at least about 70 results per hour.

[0051] The present disclosure provides an automated system for screening samples of donor blood for shipment of the donor blood or donor material for clinical use, the system comprising: a sample analysis station including a sample loading area, a sample preparation area, a nucleic acid amplification area, and a nucleic acid detection area; a processor; and a memory, the memory including instructions, when executed by the processor, for performing nucleic acid analysis on one or more samples of the donor blood to detect one or more of a plurality of pathogens or infectious agents; and if the nucleic acid analysis results in a determination of a predetermined level of nucleic acid from each of the plurality of pathogens or infectious agents, the system indicates shipment of the donor blood or donor material for clinical use, the automated system having a footprint of 1 m 2 The present invention provides an automated system that generates at least about 140 results per hour per search.

[0052] The present disclosure further provides an automated system for screening one or more samples of donor blood for shipment of the donor blood or materials from a donor for clinical use, the automated system comprising: a sample analysis station including a sample loading area, a sample preparation area, a nucleic acid amplification area, and a nucleic acid detection area; a processor; and a memory, the memory including instructions, when executed by the processor, for performing nucleic acid analysis on the one or more samples of donor blood to detect one or more of a plurality of pathogens or infectious agents; if a predetermined level of nucleic acid from each of the plurality of pathogens or infectious agents based on the nucleic acid analysis is determined, the system indicates shipment of the donor blood or donor materials for clinical use; the system is configured to screen a plurality of samples of donor blood for shipment of the donor blood or donor materials for clinical use, and the nucleic acid analysis is performed in the absence of batch processing of the plurality of samples.

[0053] In certain embodiments, the nucleic acid analysis includes optical detection of the presence of nucleic acid from at least one of the multiple pathogens or infectious agents. In certain embodiments, the system is configured to optically detect the presence of nucleic acid from at least one of the multiple pathogens or infectious agents multiple times, for example, to quantitatively detect the presence of nucleic acid from at least one of the multiple pathogens or infectious agents. In certain embodiments, the nucleic acid analysis includes a nucleic acid amplification reaction. In certain embodiments, the nucleic acid amplification reaction is an isothermal reaction. In certain embodiments, the isothermal reaction is recombinase polymerase amplification. In certain embodiments, the nucleic acid analysis includes digital detection of the presence of nucleic acid from at least one of the multiple pathogens or infectious agents.

[0054] In certain embodiments, the system is configured to digitally detect the presence of nucleic acid from at least one of a plurality of pathogens or infectious agents multiple times, e.g., to quantitatively detect the presence of nucleic acid from at least one of a plurality of pathogens or infectious agents. In certain embodiments, the nucleic acid analysis includes simultaneously contacting the sample with a sample lysis buffer and, optionally, a protease. In certain embodiments, the isothermal reaction is a recombinase polymerase amplification. In certain embodiments, the isothermal reaction is a nicking enzyme amplification reaction.

[0055] In certain embodiments, the plurality of pathogens or infectious agents is selected from the group consisting of SARS-CoV-2 (COVID-19), HIV-1, HIV-2, HBV, HCV, CMV, parvovirus B19, HAV, chlamydia, gonorrhea, WNV, Zika virus, dengue virus, chikungunya virus, influenza, babesia, malaria, Ustu virus, and HEV. In certain embodiments, the plurality of pathogens or infectious agents is HIV-1, HIV-2, HCV, and HBV. In certain embodiments, the plurality of pathogens or infectious agents is HIV-1, HIV-2, HCV, HBV, and WNV. In certain embodiments, the plurality of pathogens or infectious agents is Zika virus and WNV.

[0056] In certain embodiments, the multiple pathogens or infectious agents are chikungunya virus and dengue virus. In certain embodiments, the multiple pathogens or infectious agents are Zika virus, WNV, chikungunya virus, and dengue virus. In certain embodiments, the multiple pathogens or infectious agents are babesia and malaria. In certain embodiments, the multiple pathogens or infectious agents are parvovirus B19 and HAV.

[0057] In certain embodiments, the plurality of pathogens or infectious agents are HIV-1, HIV-2, HCV, HBV, WNV, and Zika virus. In certain embodiments, the plurality of pathogens or infectious agents are HIV-1, HIV-2, HCV, HBV, WNV, Zika virus, and Chikungunya virus. In certain embodiments, the plurality of pathogens or infectious agents are HIV-1, HIV-2, HCV, HBV, WNV, Zika virus, Chikungunya virus, and Dengue virus. In certain embodiments, the plurality of pathogens or infectious agents are HIV-1, HIV-2, HCV, HBV, WNV, Zika virus, Chikungunya virus, Dengue virus, and Babesia. In certain embodiments, the plurality of pathogens or infectious agents are HIV-1, HIV-2, HCV, HBV, WNV, Zika virus, Chikungunya virus, Dengue virus, Babesia, and malaria. In certain embodiments, the plurality of pathogens or infectious agents are HIV-1, HIV-2, HCV, HBV, WNV, Zika virus, chikungunya virus, dengue virus, babesia, malaria, and parvovirus B19. In certain embodiments, the plurality of pathogens or infectious agents are HIV-1, HIV-2, HCV, HBV, WNV, Zika virus, chikungunya virus, dengue virus, babesia, malaria, parvovirus B19, and HAV.

[0058] In certain embodiments, the multiple pathogens or infectious agents are HIV-1, HIV-2, HCV, HBV, WNV, Zika virus, Chikungunya virus, Dengue virus, Babesia, malaria, Parvovirus B19, HAV, and HEV. In certain embodiments, the multiple pathogens or infectious agents are HIV-1, HIV-2, HCV, HBV, and Parvovirus B19. In certain embodiments, the multiple pathogens or infectious agents are HIV-1, HIV-2, HCV, HBV, Parvovirus B19, and HAV.

[0059] In certain embodiments, the plurality of pathogens or infectious agents are HIV-1, HIV-2, HCV, HBV, and Babesia. In certain embodiments, the plurality of pathogens or infectious agents are HIV-1, HIV-2, HCV, HBV, and HAV. In certain embodiments, the plurality of pathogens or infectious agents are HIV-1, HIV-2, HCV, HBV, and HEV. In certain embodiments, the plurality of pathogens or infectious agents are HIV-1, HIV-2, HCV, HBV, and Zika virus. In certain embodiments, the plurality of pathogens or infectious agents are HIV-1, HIV-2, HCV, HBV, Zika virus, and Dengue virus.

[0060] In certain embodiments, the plurality of pathogens or infectious agents are HIV-1, HIV-2, HCV, HBV, Zika virus, dengue virus, and chikungunya virus. In certain embodiments, the plurality of pathogens or infectious agents are HIV-1, HIV-2, HCV, HBV, Zika virus, dengue virus, and WNV. In certain embodiments, the plurality of pathogens or infectious agents are HIV-1, HIV-2, HCV, HBV, Zika virus, WNV, and chikungunya virus. In certain embodiments, the plurality of pathogens or infectious agents are HIV-1, HIV-2, HCV, HBV, Zika virus, WNV, dengue virus, and chikungunya virus.

[0061] In certain embodiments, the multiple pathogens or infectious agents are HIV-1, HIV-2, HCV, HBV, and malaria. In certain embodiments, the multiple pathogens or infectious agents are HIV-1, HIV-2, HCV, HBV, malaria, and Babesia. In certain embodiments, the multiple pathogens or infectious agents are HIV-1, HIV-2, HCV, HBV, and dengue virus. In certain embodiments, the multiple pathogens or infectious agents are HIV-1, HIV-2, HCV, HBV, dengue virus, and chikungunya virus. In certain embodiments, the multiple pathogens or infectious agents are HIV-1, HIV-2, HCV, HBV, dengue virus, WNV, and chikungunya virus.

[0062] In certain embodiments, the plurality of pathogens or infectious agents are HIV-1, HIV-2, HCV, HBV, and chikungunya virus. In certain embodiments, the plurality of pathogens or infectious agents are HIV-1, HIV-2, HCV, HBV, chikungunya virus, and Zika virus. In certain embodiments, the plurality of pathogens or infectious agents are HIV-1, HIV-2, HCV, HBV, chikungunya virus, Zika virus, and WNV. In certain embodiments, the plurality of pathogens or infectious agents are HIV-1, HIV-2, HCV, HBV, WNV, and dengue virus. In certain embodiments, the plurality of pathogens or infectious agents are HIV-1, HIV-2, HCV, HBV, chikungunya virus, Zika virus, and WNV.

[0063] In certain embodiments, the plurality of pathogens or infectious agents are HIV-1, HIV-2, HCV, and HBV; and the nucleic acid analysis comprises multiplex analysis of HIV-1 and HIV-2. In certain embodiments, the plurality of pathogens or infectious agents are HIV-1, HIV-2, HCV, and HBV; and the nucleic acid analysis comprises multiplex analysis of HIV-1, HIV-2, and HCV. In certain embodiments, the plurality of pathogens or infectious agents are HIV-1, HIV-2, HCV, and HBV; and the nucleic acid analysis comprises multiplex analysis of HIV-1, HIV-2, and HBV. In certain embodiments, the plurality of pathogens or infectious agents are HIV-1, HIV-2, HCV, and HBV; and the nucleic acid analysis comprises multiplex analysis of HCV and HBV. In certain embodiments, the multiple pathogens or infectious agents are HIV-1, HIV-2, HCV, and HBV; the nucleic acid analysis includes multiplex analysis of HIV-1 and HIV-2; and the nucleic acid analysis includes multiplex analysis of HCV and HBV.

[0064] In certain embodiments, the multiple pathogens or infectious agents are HIV-1, HIV-2, HCV, and HBV; and the nucleic acid analysis comprises multiplex analysis of HIV-1, HIV-2, HCV, and HBV. In certain embodiments, the multiple pathogens or infectious agents comprise Zika virus, WNV, chikungunya virus, and dengue virus; and the nucleic acid analysis comprises multiplex analysis of Zika virus and WNV. In certain embodiments, the multiple pathogens or infectious agents comprise Zika virus, WNV, chikungunya virus, and dengue virus; and the nucleic acid analysis comprises multiplex analysis of chikungunya virus and dengue virus. In certain embodiments, the multiple pathogens or infectious agents comprise Zika virus, WNV, chikungunya virus, and dengue virus; and the nucleic acid analysis comprises multiplex analysis of Zika virus and WNV; and the nucleic acid analysis comprises multiplex analysis of chikungunya virus and dengue virus.

[0065] In certain embodiments, the plurality of pathogens or infectious agents comprises Zika virus, WNV, chikungunya virus, and dengue virus; the nucleic acid analysis comprises multiplex analysis of chikungunya virus and WNV; the nucleic acid analysis comprises multiplex analysis of Zika virus and dengue virus. In certain embodiments, the plurality of pathogens or infectious agents comprises Zika virus, WNV, chikungunya virus, and dengue virus; the nucleic acid analysis comprises multiplex analysis of Zika virus and dengue virus; the nucleic acid analysis comprises multiplex analysis of chikungunya virus and WNV. In certain embodiments, the plurality of pathogens or infectious agents comprises Babesia and malaria; the nucleic acid analysis comprises multiplex analysis of Babesia and malaria. In certain embodiments, the plurality of pathogens or infectious agents comprises Parvovirus B19 and HAV; the nucleic acid analysis comprises multiplex analysis of Parvovirus B19 and HAV.

[0066] In certain embodiments, nucleic acid analysis involves contacting a sample with CuTi-coated microparticles, for example during a sample preparation process. In certain embodiments, nucleic acid analysis involves contacting a sample with a plurality of microparticles and translating the microparticles across a surface via magnetic force. In certain embodiments, nucleic acid analysis involves purifying nucleic acids from a sample of donor blood; dividing the purified nucleic acids into a plurality of fractions; and retaining at least one fraction for further screening.

[0067] In certain embodiments, the plurality of pathogens or infectious agents and predetermined levels include SARS-CoV-2 (COVID-19) at a predetermined level of at least 1-50 copies / mL; HIV-1 at a predetermined level of at least 1-50 copies / mL; HIV-2 at a predetermined level of at least 1-20 IU / mL; HBV at a predetermined level of at least 1-10 IU / mL; HCV at a predetermined level of at least 1-50 IU / mL; CMV at a predetermined level of at least 10-50 IU / mL; Parvovirus B19 at a predetermined level of at least 1-40 IU / mL; HAV at a predetermined level of at least 1-10 IU / mL; chlamydia at a predetermined level; gonorrhea at a predetermined level of at least 100-500 copies / mL; WNV at a predetermined level of at least 1-50 copies / mL; Zika virus at a predetermined level of at least 1-50 copies / mL; dengue virus at a predetermined level of at least 1-50 copies / mL; chikungunya virus at a predetermined level of at least 1-50 copies / mL; influenza at a predetermined level of at least 10-500 copies / mL; Babesia at a predetermined level of at least 1-20 copies / mL; malaria at a predetermined level of at least 1-50 copies / mL; and HEV at a predetermined level of at least 1-20 IU / mL.

[0068] In certain embodiments, the plurality of pathogens or infectious agents are HIV-1, HIV-2, HCV, and HBV; the predetermined levels are at least 1-50 copies / mL of HIV-1; at least 1-20 IU / mL of HIV-2; at least 1-50 IU / mL of HCV; and at least 1-10 IU / mL of HBV. In certain embodiments, the plurality of pathogens or infectious agents are HIV-1, HIV-2, HCV, HBV, and WNV; the predetermined levels are at least 1-50 copies / mL of HIV-1; at least 1-20 IU / mL of HIV-2; at least 1-50 IU / mL of HCV; at least 1-10 IU / mL of HBV; and at least 1-50 copies / mL of WNV. In certain embodiments, the plurality of pathogens or infectious agents are Zika virus and WNV; the predetermined levels are at least 1-50 copies / mL of Zika virus and at least 1-50 copies / mL of WNV. In certain embodiments, the plurality of pathogens or infectious agents are chikungunya virus and dengue virus; the predetermined levels are at least 1-50 copies / mL of chikungunya virus; and at least 1-50 copies / mL of dengue virus. In certain embodiments, the plurality of pathogens or infectious agents are Zika virus, WNV, chikungunya virus, and dengue virus; the predetermined levels are at least 1-50 copies / mL of Zika virus; at least 1-50 copies / mL of WNV; at least 1-50 copies / mL of chikungunya virus; and at least 1-50 copies / mL of dengue virus.

[0069] In certain embodiments, the plurality of pathogens or infectious agents are Babesia and Malaria; the predetermined levels are at least 1-20 copies / mL of Babesia; and at least 1-50 copies / mL of Malaria. In certain embodiments, the plurality of pathogens or infectious agents are Parvovirus B19 and HAV; the predetermined levels are at least 1-40 IU / mL of Parvovirus B19; and at least 1-10 IU / mL of HAV. In certain embodiments, the plurality of pathogens or infectious agents are HIV-1, HIV-2, HCV, HBV, WNV, and Zika virus; the predetermined levels are at least 1-50 copies / mL of HIV-1; at least 1-20 IU / mL of HIV-2; at least 1-50 IU / mL of HCV; at least 1-10 IU / mL of HBV; at least 1-50 copies / mL of WNV; and at least 1-50 copies / mL of Zika virus. In certain embodiments, the multiple pathogens or infectious agents are HIV-1, HIV-2, HCV, HBV, WNV, Zika virus, and Chikungunya virus, and the predetermined levels are at least 1-50 copies / mL of HIV-1; at least 1-20 IU / mL of HIV-2; at least 1-50 IU / mL of HCV; at least 1-10 IU / mL of HBV; at least 1-50 copies / mL of WNV; at least 1-50 copies / mL of Zika virus; and at least 1-50 copies / mL of Chikungunya virus. In certain embodiments, the plurality of pathogens or infectious agents are HIV-1, HIV-2, HCV, HBV, WNV, Zika virus, Chikungunya virus, and Dengue virus; and the predetermined levels are at least 1-50 copies / mL of HIV-1; at least 1-20 IU / mL of HIV-2; at least 1-50 IU / mL of HCV; at least 1-10 IU / mL of HBV; at least 1-50 copies / mL of WNV; at least 1-50 copies / mL of Zika virus; at least 1-50 copies / mL of Chikungunya virus; and at least 1-50 copies / mL of Dengue virus.In certain embodiments, the plurality of pathogens or infectious agents are HIV-1, HIV-2, HCV, HBV, WNV, Zika virus, Chikungunya virus, Dengue virus, and Babesia; and the predetermined levels are at least 1-50 copies / mL of HIV-1; at least 1-20 IU / mL of HIV-2; at least 1-50 IU / mL of HCV; at least 1-10 IU / mL of HBV; at least 1-50 copies / mL of WNV; at least 1-50 copies / mL of Zika virus; at least 1-50 copies / mL of Chikungunya virus; at least 1-50 copies / mL of Dengue virus; and at least 1-20 copies / mL of Babesia.

[0070] In certain embodiments, the plurality of pathogens or infectious agents are HIV-1, HIV-2, HCV, HBV, WNV, Zika virus, Chikungunya virus, Dengue virus, Babesia, and Malaria; and the predetermined levels are at least 1-50 copies / mL of HIV-1; at least 1-20 IU / mL of HIV-2; at least 1-50 IU / mL of HCV; at least 1-10 IU / mL of HBV; at least 1-50 copies / mL of WNV; at least 1-50 copies / mL of Zika virus; at least 1-50 copies / mL of Chikungunya virus; at least 1-50 copies / mL of Dengue virus; at least 1-20 copies / mL of Babesia; and at least 1-50 copies / mL of Malaria. In certain embodiments, the plurality of pathogens or infectious agents are HIV-1, HIV-2, HCV, HBV, WNV, Zika virus, Chikungunya virus, Dengue virus, Babesia, malaria, and Parvovirus B19; and the predetermined levels are at least 1-50 copies / mL of HIV-1; at least 1-20 IU / mL of HIV-2; at least 1-50 IU / mL of HCV; at least 1-10 IU / mL of HBV; at least 1-50 copies / mL of WNV; at least 1-50 copies / mL of Zika virus; at least 1-50 copies / mL of Chikungunya virus; at least 1-50 copies / mL of Dengue virus; at least 1-20 copies / mL of Babesia; at least 1-50 copies / mL of Malaria; and at least 1-40 IU / mL of Parvovirus B19.In certain embodiments, the plurality of pathogens or infectious agents are HIV-1, HIV-2, HCV, HBV, WNV, Zika virus, Chikungunya virus, Dengue virus, Babesia, malaria, Parvovirus B19, and HAV; and the predetermined levels are at least 1-50 copies / mL of HIV-1; at least 1-20 IU / mL of HIV-2; at least 1-50 IU / mL of HCV; at least 1-10 IU / mL of HBV; at least 1-50 copies / mL of WNV; at least 1-50 copies / mL of Zika virus; at least 1-50 copies / mL of Chikungunya virus; at least 1-50 copies / mL of Dengue virus; at least 1-20 copies / mL of Babesia; at least 1-50 copies / mL of Malaria; at least 1-40 IU / mL of Parvovirus B19; and at least 1-10 IU / mL of HAV. In certain embodiments, the plurality of pathogens or infectious agents are HIV-1, HIV-2, HCV, HBV, WNV, Zika virus, Chikungunya virus, Dengue virus, Babesia, malaria, Parvovirus B19, HAV, and HEV; and the predetermined levels are at least 1-50 copies / mL of HIV-1; at least 1-20 IU / mL of HIV-2; at least 1-50 IU / mL of HCV; at least 1-10 IU / mL of HBV; and at least In certain embodiments, the plurality of pathogens or infectious agents are HIV-1, HIV-2, HCV, HBV, and parvovirus B19; and the predetermined levels are at least 1-50 copies / mL of HIV-1, at least 1-20 IU / mL of HIV-2, at least 1-50 IU / mL of HCV, at least 1-10 IU / mL of HBV, and at least 1-40 IU / mL of parvovirus B19.

[0071] In certain embodiments, the plurality of pathogens or infectious agents are HIV-1, HIV-2, HCV, HBV, parvovirus B19, and HAV; the predetermined levels are at least 1-50 copies / mL of HIV-1, at least 1-20 IU / mL of HIV-2, at least 1-50 IU / mL of HCV, at least 1-10 IU / mL of HBV, at least 1-40 IU / mL of parvovirus B19, and at least 1-10 IU / mL of HAV. In certain embodiments, the plurality of pathogens or infectious agents are HIV-1, HIV-2, HCV, HBV, and Babesia; the predetermined levels are at least 1-50 copies / mL of HIV-1, at least 1-20 IU / mL of HIV-2, at least 1-50 IU / mL of HCV, at least 1-10 IU / mL of HBV, and at least 1-20 copies / mL of Babesia. In certain embodiments, the plurality of pathogens or infectious agents are HIV-1, HIV-2, HCV, HBV, and HAV; the predetermined levels are at least 1-50 copies / mL of HIV-1, at least 1-20 IU / mL of HIV-2, at least 1-50 IU / mL of HCV, at least 1-10 IU / mL of HBV, and at least 1-10 IU / mL of HAV. In certain embodiments, the plurality of pathogens or infectious agents are HIV-1, HIV-2, HCV, HBV, and HEV; the predetermined levels are at least 1-50 copies / mL of HIV-1, at least 1-20 IU / mL of HIV-2, at least 1-50 IU / mL of HCV, at least 1-10 IU / mL of HBV, and at least 1-20 IU / mL of HEV. In certain embodiments, the multiple pathogens or infectious agents are HIV-1, HIV-2, HCV, HBV, and Zika virus; and the predetermined levels are at least 1-50 copies / mL of HIV-1; at least 1-20 IU / mL of HIV-2; at least 1-50 IU / mL of HCV; at least 1-10 IU / mL of HBV; and at least 1-50 copies / mL of Zika virus.In certain embodiments, the multiple pathogens or infectious agents are HIV-1, HIV-2, HCV, HBV, Zika virus, and Dengue virus; and the predetermined levels are at least 1-50 copies / mL of HIV-1; at least 1-20 IU / mL of HIV-2; at least 1-50 IU / mL of HCV; at least 1-10 IU / mL of HBV; at least 1-50 copies / mL of Zika virus; and at least 1-50 copies / mL of Dengue virus.

[0072] In certain embodiments, the plurality of pathogens or infectious agents are HIV-1, HIV-2, HCV, HBV, Zika virus, dengue virus, and chikungunya virus; and the predetermined levels are at least 1-50 copies / mL of HIV-1; at least 1-20 IU / mL of HIV-2; at least 1-50 IU / mL of HCV; at least 1-10 IU / mL of HBV; at least 1-50 copies / mL of Zika virus; at least 1-50 copies / mL of dengue virus; and at least 1-50 copies / mL of chikungunya virus. In certain embodiments, the plurality of pathogens or infectious agents are HIV-1, HIV-2, HCV, HBV, Zika virus, dengue virus, and WNV; and the predetermined levels are at least 1-50 copies / mL of HIV-1; at least 1-20 IU / mL of HIV-2; at least 1-50 IU / mL of HCV; at least 1-10 IU / mL of HBV; at least 1-50 copies / mL of Zika virus; at least 1-50 copies / mL of dengue virus; and at least 1-50 copies / mL of WNV. In certain embodiments, the plurality of pathogens or infectious agents are HIV-1, HIV-2, HCV, HBV, Zika virus, WNV, and Chikungunya virus; and the predetermined levels are at least 1-50 copies / mL of HIV-1; at least 1-20 IU / mL of HIV-2; at least 1-50 IU / mL of HCV; at least 1-10 IU / mL of HBV; at least 1-50 copies / mL of Zika virus; at least 1-50 copies / mL of WNV; and at least 1-50 copies / mL of Chikungunya virus. In certain embodiments, the plurality of pathogens or infectious agents are HIV-1, HIV-2, HCV, HBV, Zika virus, WNV, dengue virus, and chikungunya virus; and the predetermined levels are at least 1-50 copies / mL of HIV-1; at least 1-20 IU / mL of HIV-2; at least 1-50 IU / mL of HCV; at least 1-10 IU / mL of HBV; at least 1-50 copies / mL of Zika virus; at least 1-50 copies / mL of WNV; at least 1-50 copies / mL of dengue virus; and at least 1-50 copies / mL of Chikungunya virus.In certain embodiments, the plurality of pathogens or infectious agents are HIV-1, HIV-2, HCV, HBV, and malaria; the predetermined levels are at least 1-50 copies / mL of HIV-1, at least 1-20 IU / mL of HIV-2, at least 1-50 IU / mL of HCV, at least 1-10 IU / mL of HBV, and at least 1-50 copies / mL of malaria. In certain embodiments, the plurality of pathogens or infectious agents are HIV-1, HIV-2, HCV, HBV, malaria, and babesia; the predetermined levels are at least 1-50 copies / mL of HIV-1, at least 1-20 IU / mL of HIV-2, at least 1-50 IU / mL of HCV, at least 1-10 IU / mL of HBV, at least 1-50 copies / mL of malaria, and at least 1-20 copies / mL of babesia.

[0073] In certain embodiments, the plurality of pathogens or infectious agents are HIV-1, HIV-2, HCV, HBV, and dengue virus; the predetermined levels are at least 1-50 copies / mL of HIV-1, at least 1-20 IU / mL of HIV-2, at least 1-50 IU / mL of HCV, at least 1-10 IU / mL of HBV, and at least 1-50 copies / mL of dengue virus. In certain embodiments, the plurality of pathogens or infectious agents are HIV-1, HIV-2, HCV, HBV, dengue virus, and chikungunya virus; the predetermined levels are at least 1-50 copies / mL of HIV-1, at least 1-20 IU / mL of HIV-2, at least 1-50 IU / mL of HCV, at least 1-10 IU / mL of HBV, at least 1-50 copies / mL of dengue virus, and at least 1-50 copies / mL of chikungunya virus. In certain embodiments, the multiple pathogens or infectious agents are HIV-1, HIV-2, HCV, HBV, dengue virus, WNV, and chikungunya virus; and the predetermined levels are at least 1-50 copies / mL of HIV-1; at least 1-20 IU / mL of HIV-2; at least 1-50 IU / mL of HCV; at least 1-10 IU / mL of HBV; at least 1-50 copies / mL of dengue virus; at least 1-50 copies / mL of WNV; and at least 1-50 copies / mL of chikungunya virus. In certain embodiments, the multiple pathogens or infectious agents are HIV-1, HIV-2, HCV, HBV, and Chikungunya virus; and the predetermined levels are at least 1-50 copies / mL of HIV-1; at least 1-20 IU / mL of HIV-2; at least 1-50 IU / mL of HCV; at least 1-10 IU / mL of HBV; and at least 1-50 copies / mL of Chikungunya virus.In certain embodiments, the plurality of pathogens or infectious agents are HIV-1, HIV-2, HCV, HBV, Chikungunya virus, and Zika virus; and the predetermined levels are at least 1-50 copies / mL of HIV-1; at least 1-20 IU / mL of HIV-2; at least 1-50 IU / mL of HCV; at least 1-10 IU / mL of HBV; at least 1-50 copies / mL of Chikungunya virus; and at least 1-50 copies / mL of Zika virus.

[0074] In certain embodiments, the plurality of pathogens or infectious agents are HIV-1, HIV-2, HCV, HBV, Chikungunya virus, Zika virus, and WNV; and the predetermined levels are at least 1-50 copies / mL of HIV-1; at least 1-20 IU / mL of HIV-2; at least 1-50 IU / mL of HCV; at least 1-10 IU / mL of HBV; at least 1-50 copies / mL of Chikungunya virus; at least 1-50 copies / mL of Zika virus; and at least 1-50 copies / mL of WNV. In certain embodiments, the plurality of pathogens or infectious agents are HIV-1, HIV-2, HCV, HBV, WNV, and dengue virus; the predetermined levels are at least 1-50 copies / mL of HIV-1, at least 1-20 IU / mL of HIV-2, at least 1-50 IU / mL of HCV, at least 1-10 IU / mL of HBV, at least 1-50 copies / mL of WNV, and at least 1-50 copies / mL of dengue virus. In certain embodiments, the plurality of pathogens or infectious agents are HIV-1, HIV-2, HCV, and HBV; the nucleic acid analysis includes multiplex analysis of HIV-1 and HIV-2; the predetermined levels are at least 1-50 copies / mL of HIV-1, at least 1-20 IU / mL of HIV-2, at least 1-50 IU / mL of HCV, and at least 1-10 IU / mL of HBV. In certain embodiments, the plurality of pathogens or infectious agents are HIV-1, HIV-2, HCV, and HBV; the nucleic acid analysis comprises multiplex analysis of HIV-1, HIV-2, and HCV; and the predetermined levels are at least 1-50 copies / mL of HIV-1, at least 1-20 IU / mL of HIV-2, at least 1-50 IU / mL of HCV, and at least 1-10 IU / mL of HBV. In certain embodiments, the plurality of pathogens or infectious agents are HIV-1, HIV-2, HCV, and HBV; the nucleic acid analysis comprises multiplex analysis of HIV-1, HIV-2, and HBV; and the predetermined levels are at least 1-50 copies / mL of HIV-1, at least 1-20 IU / mL of HIV-2, at least 1-50 IU / mL of HCV, and at least 1-10 IU / mL of HBV.In certain embodiments, the multiple pathogens or infectious agents are HIV-1, HIV-2, HCV, and HBV; the nucleic acid analysis includes multiplex analysis of HCV and HBV; and the predetermined levels are at least 1-50 copies / mL of HIV-1; at least 1-20 IU / mL of HIV-2; at least 1-50 IU / mL of HCV; and at least 1-10 IU / mL of HBV.

[0075] In certain embodiments, the plurality of pathogens or infectious agents are HIV-1, HIV-2, HCV, and HBV; the nucleic acid analysis comprises multiplex analysis of HIV-1 and HIV-2; the nucleic acid analysis comprises multiplex analysis of HCV and HBV; and the predetermined levels are at least 1-50 copies / mL of HIV-1, at least 1-20 IU / mL of HIV-2, at least 1-50 IU / mL of HCV, and at least 1-10 IU / mL of HBV. In certain embodiments, the plurality of pathogens or infectious agents are HIV-1, HIV-2, HCV, and HBV; the nucleic acid analysis comprises multiplex analysis of HIV-1, HIV-2, HCV, and HBV; and the predetermined levels are at least 1-50 copies / mL of HIV-1, at least 1-20 IU / mL of HIV-2, at least 1-50 IU / mL of HCV, and at least 1-10 IU / mL of HBV. In certain embodiments, the plurality of pathogens or infectious agents are Zika virus, WNV, chikungunya virus, and dengue virus; the nucleic acid analysis comprises multiplex analysis of Zika virus and WNV; and the predetermined levels are at least 1-50 copies / mL of Zika virus; at least 1-50 copies / mL of WNV; at least 1-50 copies / mL of chikungunya virus; and at least 1-50 copies / mL of dengue virus. In certain embodiments, the plurality of pathogens or infectious agents are Zika virus, WNV, chikungunya virus, and dengue virus; the nucleic acid analysis comprises multiplex analysis of chikungunya virus and dengue virus; and the predetermined levels are at least 1-50 copies / mL of Zika virus; at least 1-50 copies / mL of WNV; at least 1-50 copies / mL of chikungunya virus; and at least 1-50 copies / mL of dengue virus.In certain embodiments, the multiple pathogens or infectious agents are Zika virus, WNV, Chikungunya virus, and Dengue virus; the nucleic acid analysis includes multiplex analysis of Zika virus and WNV; the nucleic acid analysis includes multiplex analysis of Chikungunya virus and Dengue virus; and the predetermined levels are at least 1-50 copies / mL of Zika virus; at least 1-50 copies / mL of WNV; at least 1-50 copies / mL of Chikungunya virus; and at least 1-50 copies / mL of Dengue virus. In certain embodiments, the multiple pathogens or infectious agents are Zika virus, WNV, Chikungunya virus, and Dengue virus; the nucleic acid analysis includes multiplex analysis of Chikungunya virus and WNV; the nucleic acid analysis includes multiplex analysis of Zika virus and Dengue virus; and the predetermined levels are at least 1-50 copies / mL of Zika virus; at least 1-50 copies / mL of WNV; at least 1-50 copies / mL of Chikungunya virus; and at least 1-50 copies / mL of Dengue virus.

[0076] In certain embodiments, the plurality of pathogens or infectious agents are Zika virus, WNV, chikungunya virus, and dengue virus; the nucleic acid analysis comprises multiplex analysis of Zika virus and dengue virus; the nucleic acid analysis comprises multiplex analysis of chikungunya virus and WNV; and the predetermined levels are at least 1-50 copies / mL of Zika virus; at least 1-50 copies / mL of WNV; at least 1-50 copies / mL of chikungunya virus; and at least 1-50 copies / mL of dengue virus. In certain embodiments, the plurality of pathogens or infectious agents comprise Babesia and malaria; the nucleic acid analysis comprises multiplex analysis of Babesia and malaria; and the predetermined levels are at least 1-50 copies / mL of malaria; and at least 1-20 copies / mL of Babesia. In certain embodiments, the multiple pathogens or infectious agents include parvovirus B19 and HAV; the nucleic acid analysis includes multiplex analysis of parvovirus B19 and HAV; and the predetermined levels are at least 1-40 IU / mL of parvovirus B19; and at least 1-10 IU / mL of HAV.

[0077] In certain embodiments, the sample of donor blood is human donor blood. In certain embodiments, the sample of donor blood is whole blood. In certain embodiments, the sample of donor blood is lysed whole blood. In certain embodiments, the sample of donor blood is serum. In certain embodiments, the sample of donor blood is plasma.

[0078] In certain embodiments, the donor blood sample is pooled. In certain embodiments, the pooled donor blood sample includes blood from two donors. In certain embodiments, the pooled donor blood sample includes blood from three donors. In certain embodiments, the pooled donor blood sample includes blood from four donors. In certain embodiments, the pooled donor blood sample includes blood from five donors. In certain embodiments, the pooled donor blood sample includes blood from six donors. In certain embodiments, the pooled donor blood sample includes blood from eight donors. In certain embodiments, the pooled donor blood sample includes blood from ten donors. In certain embodiments, the pooled donor blood sample includes blood from eighteen donors. In certain embodiments, the pooled donor blood sample includes blood from twelve donors. In certain embodiments, the pooled donor blood sample includes blood from twenty-four donors. In certain embodiments, the pooled donor blood sample includes blood from forty-eight donors. In one particular embodiment, the pooled sample of donor blood comprises blood from 96 donors.

[0079] In certain embodiments, the shipment of donor blood is for transfusion. In certain embodiments, the shipment of donor blood is for use in medicine. In certain embodiments, the shipment of donor blood is for use in therapeutic treatment. In certain embodiments, the sample of donor blood is for use as a blood donation. In certain embodiments, the clinical use is blood transfusion. In certain embodiments, the clinical use is use in medicine. In certain embodiments, the clinical use is use in therapeutic treatment.

[0080] The present disclosure provides a method for washing microparticles for nucleic acid analysis, comprising providing a washing container including a first well, a second well adjacent to the first well, and a third well adjacent to the second well, wherein a first sidewall defines a first side of the first, second, and third wells, a second sidewall opposite the first sidewall defines a second side of the first, second, and third wells, and the first and second sidewalls have inner surfaces facing the first, second, and third wells and an inner surface opposite the inner surface. a first partition separating the first well from the second well, a first sidewall being higher than the first partition, an inner surface of the first sidewall being substantially flat at least in a region extending between the first well and the second well; a second partition separating the second well from the third well, an inner surface of the second sidewall being substantially flat at least in a region extending between the second well and the third well; and introducing magnetic microparticles into the first well, a first well containing a washing solution; applying a magnetic force to capture magnetic microparticles on the inner surface of a first sidewall in the first well; translating the captured magnetic microparticles across a first partition from the first well along the inner plane of the first sidewall to a second well, where the second well contains the washing solution; removing the magnetic force to release the magnetic microparticles from the inner surface into the second well; applying a magnetic force to capture the magnetic microparticles on the inner surface of a second sidewall opposite the first sidewall, where the second sidewall is higher than the second partition; and translating the captured magnetic microparticles across the second partition from the second well along the inner plane of the second sidewall to a third well, where the third well contains the washing solution or elution solution; removing the magnetic force to release the magnetic microparticles from the inner surface of the second sidewall into the third well. In certain embodiments, the microparticle washing methods disclosed herein can be used during the sample preparation process.

[0081] In certain embodiments, the third well contains an elution solution, and the method further includes applying a magnetic force to the magnetic microparticles in the third well to capture the magnetic microparticles on the inner surface of the first sidewall, and removing the elution solution. In certain embodiments, the washing container includes a fourth well, wherein the third well contains the washing solution and the fourth well contains the elution solution, and the third and fourth wells are separated by a third partition. In certain embodiments, first and second side walls extend along the first, second, third, and fourth wells and define opposing sides of the wells, the inner surfaces of the first and second side walls face the wells, the inner surface of the first side wall is non-planar in the region extending between the second well and the third well and planar in the region extending between the third well and the fourth well, and the inner surface of the second side wall is non-planar in the region extending between the third well and the fourth well, the planar regions facilitate translational movement of captured magnetic particles across the inner surface, and the non-planar regions prevent translational movement of captured magnetic particles across the inner surface.

[0082] In certain embodiments, the method further includes applying a magnetic force to capture magnetic particles in the third well on the inner surface of the first sidewall, where the first sidewall is higher than the third partition; translating the captured magnetic microparticles from the third well along the inner plane of the first sidewall to the fourth well, beyond the third partition; and removing the magnetic force to release the magnetic microparticles from the inner surface of the first sidewall into the fourth well. In certain embodiments, the third well contains a wash solution and the fourth well contains an elution solution, and the method further includes applying a magnetic force to the magnetic microparticles in the fourth well to capture the magnetic microparticles on the inner surface of the second sidewall and removing the elution solution. In certain embodiments, the magnetic force is generated using an electromagnet. In certain embodiments, the electromagnet is disposed along the outer surfaces of the first and second sidewalls, and translating the captured magnetic microparticles includes sequentially activating and deactivating different regions of the electromagnet. In certain embodiments, the magnetic force is generated using a permanent magnet. In certain embodiments, translating the captured magnetic microparticles comprises moving a permanent magnet along an outer surface of the first or second side wall, hi certain embodiments, the non-planar inner surface comprises a notch therein, the notch extending outwardly from or within the inner surface.

[0083] The present disclosure also provides a washing container comprising: a first well adjacent to a second well and a third well adjacent to the second well; a first sidewall defining first sides of the first, second, and third wells; a second sidewall opposite the first sidewall and defining second sides of the first, second, and third wells, the first sidewall and second sidewall including an inner surface facing the first, second, and third wells and an outer surface opposite the inner surface; a first partition separating the first well from the second well; and a second partition separating the second well from the third well, wherein the first sidewall is higher than the first partition and the inner surface of the first sidewall allows magnetic microparticles captured in the first well to pass over the first partition and into the second well. a washing container having a first sidewall that is substantially planar in at least a region extending between the first well and the second well, and an inner surface of the first sidewall that is non-planar in a region extending between the second well and the third well, such that the non-planar inner surface prevents magnetic particles captured in the region of the inner surface of the second well from translating to the region of the inner surface of the third well; a second sidewall that is higher than the second partition, and an inner surface of the second sidewall that is substantially planar in at least a region extending between the second well and the third well, providing a substantially flat surface for capturing magnetic microparticles in the second well and translating the captured magnetic particles across the second partition to the third well.

[0084] In certain embodiments, the first and second wells contain a wash solution, and the third well contains a wash solution or an elution solution. In a specific embodiment, the washing container includes a fourth well adjacent to the third well and a third partition separating the third well and the fourth well, wherein a first sidewall defines a first side of the fourth well, a second sidewall defines a second side of the fourth well, the first sidewall is higher than the third partition, the inner surface of the first sidewall is substantially planar in at least a region extending between the third well and the fourth well to allow magnetic microparticles captured in the third well to translate across the third partition to the fourth well, and the inner surface of the second sidewall is non-planar in a region extending between the third well and the fourth well, such that the non-planar inner surface prevents magnetic particles captured in the region of the inner surface of the second sidewall in the third well from translating to the region of the inner surface of the second sidewall of the fourth well. In certain embodiments, the first, second, and third wells contain a wash solution. In certain embodiments, the first, second, and third wells contain a wash solution, and the fourth well contains an elution solution. In certain embodiments, the non-planar inner surface comprises a notch therein, the notch extending outwardly from the inner surface or extending within the inner surface.

[0085] The present disclosure further provides a method for screening a sample related to donor blood for shipment of donor blood or materials from a donor for clinical use, the method comprising: preparing the sample for nucleic acid analysis, comprising providing nucleic acid isolated from the sample in a volume of eluate; and dispensing the volume of eluate into at least two amplification vessels. In certain embodiments, the method can further comprise subjecting the eluate in the at least two amplification vessels to a nucleic acid amplification reaction (e.g., during an amplification and detection process); and detecting the presence or absence of nucleic acid of a pathogen or infectious agent in the eluate.

[0086] In certain embodiments, dispensing includes dispensing the volume of eluate into at least three, four, or more amplification vessels: subjecting the eluate in the amplification vessels to a nucleic acid amplification reaction (e.g., during an amplification and detection process); and detecting the presence or absence of pathogen or infectious agent nucleic acid in the eluate. In certain embodiments, equal volumes of eluate are dispensed into the amplification vessels. In certain embodiments, different volumes of eluate are dispensed into the amplification vessels. In certain embodiments, the amplification reaction in each amplification vessel is for amplifying nucleic acid present in a different pathogen or infectious agent. In certain embodiments, the amplification reaction in each amplification vessel is for amplifying nucleic acid present in multiple different pathogens or infectious agents. In certain embodiments, the amplification reaction comprises an isothermal reaction. In certain embodiments, the isothermal reaction is a recombinase polymerase amplification. In certain embodiments, the isothermal reaction is a nicking enzyme amplification reaction.

[0087] The present disclosure further provides a method for detecting a target nucleic acid in a sample. By way of example and not limitation, the method can include: preparing nucleic acid from a sample for an isothermal amplification reaction; amplifying the target nucleic acid by the isothermal amplification reaction; and determining the amount of the target nucleic acid amplified in the reaction, and / or determining the presence or absence of the target nucleic acid amplified in the reaction, wherein the sample preparation, the amplification reaction, and the determination of the amount, presence or absence of the target nucleic acid amplified in the amplification reaction are completed in less than about 60 minutes, for example, less than about 20 minutes. In certain embodiments, the present disclosure provides a method for detecting a target nucleic acid of a pathogen or infectious agent in a sample, comprising preparing nucleic acid from the sample for an isothermal amplification reaction; amplifying the target nucleic acid by the isothermal amplification reaction; and determining the amount of the target nucleic acid amplified in the reaction and / or determining the presence or absence of the target nucleic acid amplified in the reaction, wherein the sample preparation, the amplification reaction, and the determination of the amount, presence, or absence of the target nucleic acid amplified in the amplification reaction are completed in less than about 60 minutes, e.g., in less than about 20 minutes.

[0088] In certain embodiments, determining the amount of the target nucleic acid comprises optically detecting a fluorescent signal corresponding to the amount of the target nucleic acid multiple times during the amplification reaction. In certain embodiments, determining the presence or absence of the target nucleic acid comprises optically detecting a fluorescent signal corresponding to the target nucleic acid multiple times during the amplification reaction. In certain embodiments, the multiple times comprise optically detecting the signal at predetermined intervals of about every 20 seconds or about every 30 seconds. In certain embodiments, the multiple optical detections of the fluorescent signal are performed over a period of about 12 minutes. In certain embodiments, the method is for screening a sample related to donor blood for shipping the donor blood for clinical use. In certain embodiments, sample preparation, the amplification reaction, and determination of the amount, presence, or absence of the target nucleic acid amplified in the amplification reaction are completed in about 15 minutes to about 60 minutes, e.g., about 20 minutes to about 60 minutes. In certain embodiments, the target nucleic acid comprises nucleic acid from parvovirus B19 and hepatitis A virus (HAV). In certain embodiments, the target nucleic acid comprises nucleic acid from multiple pathogens or infectious agents. In certain embodiments, the plurality of pathogens or infectious agents comprises HIV-1, HIV-2, HBV, and HCV. In certain embodiments, the plurality of pathogens or infectious agents comprises HIV-1 and HBV. In certain embodiments, the plurality of pathogens or infectious agents comprises HIV-2 and HCV.

[0089] The present disclosure further provides methods for screening samples of donor blood for shipping of the donor material for clinical use. In certain embodiments, the donor material can be, for example, blood products such as whole blood, platelets, red blood cells, and plasma. In certain embodiments, the donor material can be, for example, tissues, organs, vaccines, cells, gene therapies, and recombinant therapeutic proteins.

[0090] According to another aspect of the disclosed subject matter, the methods and systems of the present disclosure can be used to analyze biological samples other than donor blood. For example, in certain embodiments, the biological sample is a bodily secretion such as, for example, saliva or oral fluid, sweat, tears, mucus, urine, lymph, cerebrospinal fluid, interstitial fluid, bronchoalveolar lavage fluid, or any other sample suitable for analysis using the methods and techniques described herein. [Brief explanation of the drawings]

[0091] [Figure 1]

[0013] Figure 1 illustrates an exemplary HTNAT sample analysis process according to certain embodiments of the disclosed subject matter, as well as exemplary advantages associated with certain embodiments of the disclosed sample preparation, amplification, and detection strategies.

[0014] Figure 1 illustrates an exemplary HTNAT sample analysis process according to certain embodiments of the disclosed subject matter, including three processes: a sample preparation process, an amplification process, and a detection process. In certain embodiments, the amplification process and the detection process are performed simultaneously in the amplification and detection process. [Figure 2A] 1 is a diagram illustrating an exemplary automated HTNAT sample analysis process according to the disclosed subject matter, including three processes: a sample preparation process (e.g., a pre-lysis process, a sample lysis process, and a wash and elution process), an amplification process (including reagent addition), and a detection / read process. [Figure 2B] 1 is a diagram showing an exemplary HTNAT automated sample analysis process according to the disclosed subject matter;FIG. 2 is a diagram showing an exemplary HTNAT automated sample preparation protocol for processing plasma / serum or whole blood sample types according to the disclosed subject matter; [Figure 3] 1 is a diagram illustrating an exemplary HTNAT sample analysis system according to the disclosed subject matter, including three processes: a sample preparation process (e.g., a pretreatment lysis process, a sample lysis process, and a wash and elution process), an amplification process, and a detection / read process. [Figure 4]1 is a diagram showing an exemplary embodiment of a sample preparation process (direct capture and CuTi total nucleic acid capture) according to the disclosed subject matter. [Figure 5] 1 is a diagram illustrating an exemplary pre-treatment lysis process (also referred to herein as pre-lysis) (sample preparation) system according to the disclosed subject matter. [Figure 6] 1 is a diagram illustrating an exemplary sample lysis process (sample preparation) system according to the disclosed subject matter. [Figure 7] 1 is a diagram illustrating an exemplary washing process (also referred to herein as washing and elution) (sample preparation) system according to the disclosed subject matter. [Figure 8] 1 is a diagram illustrating an exemplary amplification and detection system in accordance with the disclosed subject matter. [Figure 9] 1 is a diagram showing an exemplary CuTi total nucleic acid capture process improvement (highlighted) of the disclosed subject matter. [Figure 10] 1 is a diagram illustrating an exemplary direct capture process improvement of the disclosed subject matter. [Figure 11] 1 shows exemplary results related to RPA amplification of HBV following digital detection (left) or fluorescence (right) implementation of the disclosed subject matter. [Figure 12] 1 shows exemplary results relating to the implementation of NEARHCV amplification and fluorescence detection of the disclosed subject matter. The table lists the oligonucleotides used. [Figure 13] FIG. 1 illustrates exemplary digital and optical (fluorescence-based) target detection strategies. [Figure 14] 1 shows exemplary results associated with RPA implementations of the disclosed subject matter. Purple traces represent separate replicates within the same experiment; gray traces are negative controls. The table lists the RPA oligonucleotides used. [Figure 15] 1 is a diagram of an exemplary mix and wash embodiment identifying 18 different locations where stationary electromagnet-based capture and washing of magnetic particles can be incorporated. [Figure 16]1 is a diagram of an exemplary mixing and washing embodiment identifying four pairs of positions that can incorporate stationary electromagnet-based transport of magnetic particles. [Figure 17] A shows an exemplary design of a washing vessel for use in combination with a moving permanent magnet or a fixed electromagnet, and the approximate volume of four wells of the vessel. B shows an exemplary design of a washing vessel for use in combination with a moving permanent magnet or a fixed electromagnet, and the approximate volume of four wells of the vessel. [Figure 18A] Graph of RPA of gene (ORF): VP1 of parvovirus B19 (cycles indicate fluorescence readings every 60 seconds) using various combinations of amplification and oligonucleotides. Each trace on the plot represents a different replicate. [Figure 18B] Graph of RPA of gene (ORF): VP1 of parvovirus B19 (cycles indicate fluorescence readings every 60 seconds) using various combinations of amplification and oligonucleotides. Each trace on the plot represents a different replicate. [Figure 18C] Graph of RPA of gene (ORF): NS1 of parvovirus B19 (cycles indicate fluorescence readings every 60 seconds) using various combinations of amplification and oligonucleotides. Each trace on the plot represents a different replicate. [Figure 18D] 1 is a graph of RPA of gene (ORF): NS1 of parvovirus B19 (cycles indicate fluorescence readings every 60 seconds) using various combinations of amplification and oligonucleotides. Each trace on the plot represents a different replicate. Each trace on the plot represents a different replicate. [Figure 18E] Graph of RPA of the 5' untranslated region (UTR) of a single HAV polypeptide using various combinations of amplification and oligonucleotides. Each trace on the plot represents a different replicate. [Figure 18F] Graph of RPA of the 5' untranslated region (UTR) of a single HAV polypeptide using various combinations of amplification and oligonucleotides. Each trace on the plot represents a different replicate. [Figure 19] A is a graph of RPA for various Babesia parasite species using various combinations of amplification and probe oligonucleotides as indicated. Each cycle represents a fluorescence reading every 60 seconds. B is a graph of RPA for various Babesia parasite species using various combinations of amplification and probe oligonucleotides as indicated. Each cycle represents a fluorescence reading every 60 seconds. C is a graph of RPA for various Babesia parasite species using various combinations of amplification and probe oligonucleotides as indicated. Each cycle represents a fluorescence reading every 60 seconds. D is a graph of RPA for various Babesia parasite species using various combinations of amplification and probe oligonucleotides as indicated. Each cycle represents a fluorescence reading every 60 seconds. [Figure 20] A is a graph of RPA for human immunodeficiency virus 1 (HIV-1). Each trace on the plot represents a different replication. B is a graph of RPA for human immunodeficiency virus 2 (HIV-2). Each trace on the plot represents a different replication. C is a graph of RPA for hepatitis C virus (HCV). Each trace on the plot represents a different replication. D is a graph of RPA for hepatitis B virus (HBV). Each trace on the plot represents a different replication. [Figure 21] 1 is a diagram showing an exemplary RPA amplification protocol according to the disclosed subject matter. [Figure 22] Exemplary results related to improved RNA detection facilitated by the use of selected reverse transcriptases are shown. HCV RNA amplification is used as an example. Values ​​in boxes indicate the target (HCV RNA) concentration and the reverse transcriptase used. [Figure 23](A) shows exemplary results relating to improved RPA amplification of HCV in the presence of primers for HBV, demonstrating inhibition of HCV in the presence of HBV oligonucleotides. Although not shown, HBV amplification is unaffected by this change. (B) shows exemplary results relating to improved RPA amplification of HCV in the presence of primers for HBV, demonstrating restoration of HCV amplification with a decrease in the overall concentration of HBV oligonucleotides. Although not shown, HBV amplification is unaffected by this change. [Figure 24] Figure 1 shows a graph of singleplex RPA of HIV-1 using amplification and probe oligonucleotides at or near the desired limit of detection (LOD), as indicated. Each curve represents a single replicate (n=6) at the noted target concentration. [Figure 25] Figure 1 shows a graph of singleplex RPA of HIV-2 using amplification and probe oligonucleotides at or near the desired limit of detection (LOD), as indicated. Each curve represents a single replicate (n=6) at the noted target concentration. [Figure 26] Graph of singleplex RPA of HBV using amplification and probe oligonucleotides at or near the desired limit of detection (LOD), as indicated. Each curve represents a single replicate (n=6) at the noted target concentration. [Figure 27] Graph of singleplex RPA of HCV using amplification and probe oligonucleotides at or near the desired limit of detection (LOD), as indicated. Each curve represents a single replicate (n=6) at 10 IU per reaction. [Figure 28] Figure 1 shows a graph of multiplex RPA of HIV-1, HBV, and an internal control at the indicated target levels. Note that the 1x LOD for HIV-1 is 20 copies / mL and for HBV is 5 IU / mL. The numbers in the upper left of the plot represent the number of positive replicates out of the total number tested. [Figure 29]Figure 1 shows a graph of multiplex RPA of HIV-2, HCV, and an internal control at the indicated target levels. Note that the 1x LOD for HIV-2 is 20 IU / mL and for HCV is 10 IU / mL. The numbers in the upper left of the plot represent the number of positive replicates out of the total number tested. [Figure 30] (A) Singleplex RPA of HAV using amplification and probe oligonucleotides as indicated. (B) Singleplex RPA of Parvo using amplification and probe oligonucleotides as indicated. Each cycle represents a fluorescence reading every 30 seconds. Target levels and probe fluorophores are indicated above the amplification plot. [Figure 31A] 1 is a graph of singleplex RPA of Parvo at different target nucleic acid concentrations. [Figure 31B] The data in Figure 31A are graphed to establish a standard curve showing cycle threshold (Ct) on the y-axis and logarithmic target level concentration on the x-axis (each data point represents a single replicate). [Figure 31C] The correlation between calculated viral titers on the y-axis and actual viral titers on the x-axis, as determined by the regression equation in Figure 31B, is graphed. [Figure 31D] The table is a numerical output of the data from the plots, showing acceptable linearity across the virus titers tested. [Figure 32] A shows the location of primers and probes relative to the Chikungunya virus genome. B shows a graph of singleplex RPA of Chikungunya virus using the indicated amplification primers and probe oligonucleotides, as well as target levels in copy numbers per reaction of in vitro transcribed RNA. C shows a graph of singleplex RPA of Chikungunya virus using the indicated amplification primers and probe oligonucleotides, as well as target levels in copy numbers per reaction of in vitro transcribed RNA. D lists the sequences of the indicated primers and probes in a table. [Figure 33]A is a graph of singleplex RPA of dengue virus using the conditions shown in B. B shows the conditions. C contains the primer and probe sequences shown. [Figure 34] A shows the location of primers and probes relative to the West Nile virus (WNV) genome. B is a graph of singleplex RPA of WNV using the indicated amplification primers and probe oligonucleotides. C is a graph of singleplex RPA of WNV using the indicated amplification primers and probe oligonucleotides. D is the sequence of the primers and probe. E is a graph of singleplex RPA of WNV using the indicated amplification oligonucleotides and probe oligonucleotides at different target nucleic acid concentrations. [Figure 35] A shows the location of primers and probes relative to the Zika virus genome. B is a graph of singleplex RPA of Zika virus using the indicated amplification primer and probe oligonucleotides. C is the sequence of the indicated primers and probes. [Figure 36] A contains a graph of multiplex RPA of Babesia using the indicated Babesia amplification primers and probe oligonucleotides with sequences provided in the table in C. B contains a graph of the internal control. C shows the sequences of the indicated Babesia amplification primers and probe oligonucleotides. [Figure 37] A contains a graph of multiplex RPA of malaria using the indicated malaria amplification primer and probe oligonucleotides with sequences provided in the table in C. B contains a graph of the internal control. C shows the sequences of the indicated malaria amplification primer and probe oligonucleotides. [Figure 38] A contains a graph of RPA amplification and detection of SARS-CoV-2 (COVID-19) targeting the RdRp genomic region. B contains a graph of RPA amplification and detection of SARS-CoV-2 (COVID-19) targeting the N genomic region. [Figure 39]A contains a graph of multiplex RPA for HIV-1 showing that the indicated limits of detection can be achieved using the disclosed methods within the indicated time frames. B contains a graph of multiplex RPA for HBV showing that the indicated limits of detection can be achieved using the disclosed methods within the indicated time frames. [Figure 40] A contains a graph of multiplex RPA for HIV-2 showing that the indicated limits of detection can be achieved using the disclosed methods within the indicated time frames. B contains a graph of multiplex RPA for HCV showing that the indicated limits of detection can be achieved using the disclosed methods within the indicated time frames. [Figure 41] A contains a graph of multiplex RPA for parvovirus B19 showing that the indicated limits of detection can be achieved using the methods of the present disclosure within the indicated time frames.B contains a graph of multiplex RPA for HAV showing that the indicated limits of detection can be achieved using the methods of the present disclosure within the indicated time frames. [Figure 42] A shows an exemplary embodiment of an electromagnet-based or moving permanent magnet-based sample processing and related strategies useful in connection with the system of the present disclosure. B shows an exemplary embodiment of an electromagnet-based or moving permanent magnet-based sample processing and related strategies useful in connection with the system of the present disclosure. C shows an exemplary embodiment of an electromagnet-based or moving permanent magnet-based sample processing and related strategies useful in connection with the system of the present disclosure. D shows an exemplary embodiment of an electromagnet-based or moving permanent magnet-based sample processing and related strategies useful in connection with the system of the present disclosure. [Figure 43] 1 illustrates an exemplary embodiment of a split eluate sample processing system and related strategies useful in connection with the systems of the present disclosure. [Figure 44]A contains a graph of multiplex HxVRPA results for HIV-1, showing that the indicated limits of detection can be achieved using the methods of the present disclosure. B contains a graph of multiplex HxVRPA results for HIV-2, showing that the indicated limits of detection can be achieved using the methods of the present disclosure. C contains a graph of multiplex HxVRPA results for HBV, showing that the indicated limits of detection can be achieved using the methods of the present disclosure. D contains a graph of multiplex HxVRPA results for HCV, showing that the indicated limits of detection can be achieved using the methods of the present disclosure. [Figure 45] 1 illustrates an exemplary embodiment of a sample processing cartridge and associated strategies useful in connection with the systems of the present disclosure. [Figure 46A] 1 illustrates an exemplary embodiment of a heat block and associated heating strategy useful in connection with the system of the present disclosure. [Figure 46B] 1 illustrates an exemplary embodiment of a heat block and associated heating strategy useful in connection with the system of the present disclosure. [Figure 46C] 1 illustrates an exemplary embodiment of a heat block and associated heating strategy useful in connection with the system of the present disclosure. [Figure 47] A shows an exemplary embodiment of the overall system architecture, e.g., sample handling locations, reagent locations, and associated computer processing units, as well as associated sample processing strategies useful in connection with the disclosed system. B shows an exemplary embodiment of the overall system architecture, e.g., sample handling locations, reagent locations, and associated computer processing units, as well as associated sample processing strategies useful in connection with the disclosed system. [Figure 48] 1 illustrates exemplary embodiments of sample tubes and sample tube processing strategies, such as the use of specific lanes, useful in connection with the systems of the present disclosure. [Figure 49] 1 illustrates an exemplary embodiment of a loading shelf strategy useful in connection with the system of the present disclosure. [Figure 50] 1 illustrates an exemplary embodiment of a code reader strategy useful in connection with the system of the present disclosure. [Figure 51] 1 illustrates an exemplary embodiment of a conveyor strategy useful in connection with the system of the present disclosure. [Figure 52] 1A and 1B show an exemplary embodiment of the disclosed system for a pipette tip rack loader; [Figure 53] 1 illustrates an exemplary embodiment of the system of the present disclosure with respect to the configuration of a sample preparation cartridge loading area and associated reagent containers. [Figure 54A] 1 illustrates an exemplary sample preparation cartridge transfer station and a sample preparation cartridge filling station according to one embodiment. [Figure 54B] 1 illustrates an exemplary sample preparation cartridge transfer station and a sample preparation cartridge filling station according to one embodiment. [Figure 54C] 1 illustrates an exemplary sample preparation cartridge transfer station and a sample preparation cartridge filling station according to one embodiment. [Figure 54D] 1 illustrates an exemplary sample preparation cartridge transfer station and a sample preparation cartridge filling station according to one embodiment. [Figure 54E] 1 illustrates an exemplary sample preparation cartridge transfer station and a sample preparation cartridge filling station according to one embodiment. [Figure 55A] 1 illustrates an exemplary embodiment of a system of the present disclosure for robotic handling of sample preparation cartridges. [Figure 55B] 1 illustrates an exemplary embodiment of a system of the present disclosure for robotic handling of sample preparation cartridges. [Figure 55C] 1 illustrates an exemplary embodiment of a system of the present disclosure for robotic handling of sample preparation cartridges. [Figure 56] 1 illustrates an exemplary embodiment of a system of the present disclosure for filling a sample preparation cartridge. [Figure 57] 1 illustrates an exemplary embodiment of a system of the present disclosure for storing ancillary reagents. [Figure 58] 1 illustrates an exemplary embodiment of a system of the present disclosure for storing bulk reagents. [Figure 59] 1 illustrates an exemplary embodiment of a system of the present disclosure that utilizes a magnet-based system, such as the Magtration® system, to isolate magnetic particles within a pipette tip. [Figure 60A] 1 illustrates an exemplary embodiment of the system of the present disclosure in which amplification vessels are transported to locations via trucks. [Figure 60B] 1 illustrates an exemplary embodiment of the system of the present disclosure in which amplification vessels are transported to locations via trucks. [Figure 60C] 1 illustrates an exemplary embodiment of the system of the present disclosure in which amplification vessels are transported to locations via trucks. [Figure 61] 1 illustrates an exemplary embodiment of the system of the present disclosure, in which the reader, e.g., an optical detector, is movable while the amplification vessel is stationary. [Figure 62] FIG. 1 shows a plan view of an exemplary HTNAT sample analysis system for performing sample preparation, amplification, and detection, as well as additional components associated with obtaining results from the HTNAT sample analysis system, such as sample handling, loading of consumables, and waste disposal. [Figure 63] FIG. 1 shows a perspective view of an exemplary HTNAT sample analysis system for performing sample preparation, amplification, and detection, as well as additional components associated with obtaining results from the HTNAT sample analysis system, such as sample handling, loading of consumables, and waste disposal. [Figure 64] 1 illustrates an exemplary sample transport for performing sample preparation in connection with obtaining results from an exemplary HTNAT sample analysis system. [Figure 64A] 1 shows an exemplary schematic diagram of a sample transporter for performing sample mixing and an exemplary rotational movement of the sample transporter. [Figure 64B] 64B shows an exemplary schematic diagram of the lysis tube during the rotational movement of the sample transporter shown in FIG. 64A. [Figure 64C] 64B shows an exemplary schematic diagram of the lysis tube during the rotational movement of the sample transporter shown in FIG. 64A. [Figure 65] 1 illustrates an exemplary wash and elution system for performing sample preparation in connection with obtaining results from an exemplary HTNAT sample analysis system. [Figure 66] 1 shows an exemplary amplification and detection system associated with obtaining results from an exemplary HTNAT sample analysis system. [Figure 67A] 1 illustrates exemplary embodiments of the split eluate aspect of the present disclosure, showing a no split scenario (top process path), a split on odd number of assays scenario (middle process path), and a split on even number of assays scenario (bottom process path). [Figure 67B] 1 shows exemplary embodiments of the split eluate aspect of the present disclosure. Various scenarios of assay combination and eluate splitting illustrate the advantages and utility of eluate splitting. [Figure 68A] 1 shows an exemplary processing deck with pipettors of an exemplary HTNAT sample analysis system. [Figure 68B] 1 shows an exemplary processing deck with pipettors of an exemplary HTNAT sample analysis system. [Figure 68C] 1 shows an exemplary processing deck with pipettors of an exemplary HTNAT sample analysis system. [Figure 68D] 1 shows an exemplary processing deck with pipettors of an exemplary HTNAT sample analysis system. [Figure 69A] 1A-1C illustrate exemplary embodiments of lysis tubes and transfer tips for use in an HTNAT sample analysis system, as well as exemplary stacking configurations of lysis tubes and transfer tips. [Figure 69B] 10A-10C illustrate additional exemplary embodiments of lysis tubes and transfer tips for use in the HTNAT sample analysis system, as well as exemplary stacking configurations of lysis tubes and transfer tips. [Figure 69C] FIG. 10 shows a top view of another exemplary embodiment of a lysis tube for use in the HTNAT sample analysis system. [Figure 69D] 69C taken along line AA as shown in FIG. 69C. [Figure 69E] FIG. 10 shows a top view of another exemplary embodiment of a lysis tube for use in the HTNAT sample analysis system. [Figure 69F] 69E shows a cross-sectional side view of the exemplary lysis tube shown in FIG. 69E taken along line BB as shown in FIG. 69E. [Figure 70] FIG. 68B shows an exemplary wash track of the exemplary HTNAT sample analysis system. [Figure 71] 44 illustrates an exemplary cleaning container for use in the exemplary cleaning truck of FIG. 43. [Figure 72] FIG. 68B shows an exemplary amplification and detection system for the exemplary HTNAT sample analysis system. [Figure 73] 73 shows an exemplary amplification vessel for use in the amplification and detection system of FIG. 72. [Figure 74] 1 illustrates an exemplary sample loading bay for use in an exemplary HTNAT sample analysis system. [Figure 75] 1 illustrates an exemplary embodiment of a loading bay with a sample tray rack. [Figure 76] 10 illustrates mixing of the contents of a lysis tube using the rotational action of a rotating carousel, according to one aspect of the disclosed subject matter. [Figure 77] 1 illustrates an exemplary whole blood sample processing method using systems and devices according to the disclosed subject matter. [Figure 78] 1 illustrates an exemplary system in accordance with the disclosed subject matter. [Figure 79] 1 illustrates an exemplary method in accordance with the disclosed subject matter. [Figure 80] 1 illustrates an exemplary embodiment of a cleaning vessel. [Figure 81] FIG. 68B is a top view of an exemplary sample preparation carousel of the exemplary HTNAT sample analysis system of FIGS. 68A-68D. [Figure 82]81A and 81B are schematic top views of the sample preparation carousel of FIG. 81 showing an example of pooling a first sample and a second sample into tubes on the sample preparation carousel according to one embodiment of the disclosed subject matter. [Figure 83] 81A and 81B are schematic top views of the sample preparation carousel of FIG. 81 showing an example of pooling a third sample and a fourth sample, respectively. [Figure 84A] FIG. 82 is a schematic top view of the sample preparation carousel of FIG. 81 , illustrating an exemplary pretreatment process and pooling of pretreated samples according to another embodiment of the disclosed subject matter. [Figure 84B] FIG. 82 is a schematic top view of the sample preparation carousel of FIG. 81 , illustrating an exemplary pretreatment process and pooling of pretreated samples according to another embodiment of the disclosed subject matter. [Figure 84C] FIG. 82 is a schematic top view of the sample preparation carousel of FIG. 81 , illustrating an exemplary pretreatment process and pooling of pretreated samples according to another embodiment of the disclosed subject matter. [Figure 85A] 1 illustrates an exemplary pool decomposition strategy. [Figure 85B] 1 illustrates an exemplary pool decomposition strategy. [Figure 85C] 1 illustrates an exemplary pool decomposition strategy. [Figure 85D] 1 illustrates an exemplary pool decomposition strategy. [Figure 85E] 1 illustrates an exemplary pool decomposition strategy. [Figure 85F] 1 illustrates an exemplary pool decomposition strategy. [Figure 86] A shows an exemplary throughput of an exemplary sample type and a process of an exemplary system according to the disclosed subject matter.B shows an exemplary throughput of an exemplary sample type and a process of an exemplary system according to the disclosed subject matter. [Figure 87]1A shows an exemplary method for on-board pooling of 12 samples according to one embodiment of the disclosed subject matter. 1B shows an exemplary method for on-board pooling of 12 samples according to one embodiment of the disclosed subject matter. [Figure 88] 1A shows an exemplary method for on-board pooling of 18 samples according to one embodiment of the disclosed subject matter. 1B shows an exemplary method for on-board pooling of 18 samples according to one embodiment of the disclosed subject matter. [Figure 89] 1A shows an exemplary method for on-board pooling of 24 samples according to one embodiment of the disclosed subject matter. 1B shows an exemplary method for on-board pooling of 24 samples according to one embodiment of the disclosed subject matter. [Figure 90] 1 illustrates an exemplary pretreatment process and on-board pooling of pretreated samples according to one aspect of the disclosed subject matter. DETAILED DESCRIPTION OF THE INVENTION

[0092] The subject matter disclosed herein relates to various methods and systems for rapidly detecting target nucleic acids in a sample. For example, without limitation, the subject matter disclosed herein is directed to various methods and systems for rapidly screening donor blood or donor material (e.g., whole blood, lysed whole blood, serum, or plasma) using a unique nucleic acid assay to detect one or more pathogens or infectious agents more efficiently than conventional techniques, wherein the nucleic acid assay of the disclosed subject matter is sufficiently sensitive that determining a predetermined level of nucleic acid derived from a pathogen or infectious agent indicates whether the donor blood or donor-derived material (i.e., the donor material) can be shipped for clinical use, such as for transfusion, transplantation, or incorporation into a therapeutic.

[0093] The methods and systems disclosed herein modify current processes / systems for NAT-based analysis of samples, e.g., for donor blood screening. By eliminating significant bottlenecks in blood and plasma screening caused by conventional screening assays, the disclosed methods and systems significantly modify donor blood processes, simultaneously improving efficiency and safety. For example, current NAT-based screening can have assay times several hours longer than serology-based screening (e.g., 3.5 hours of time to first result) and typically requires sample pooling and batch processing to achieve meaningful throughput, which can introduce inefficiencies into traditional blood screening laboratory operations. In contrast, the methods and systems disclosed herein reduce the time to first results for NAT-based screening from several hours to minutes, while maintaining or even significantly reducing equipment size to achieve improved overall sample throughput. Importantly, the methods and systems described herein not only provide rapid screening of donor blood, but also reduce the need for blood testing centers to perform sample pooling and restrictive batch processing to achieve the throughput required to efficiently monitor donor blood.

[0094] In addition to the increased throughput provided by the unique NAT-based screening developments described herein for screening donor blood, the disclosed methods and systems, e.g., the sample preparation, amplification, and detection deployments disclosed herein, also enable highly flexible sample processing. For example, the methods and systems described herein provide the ability to interrupt workflow to process priority samples and to modify a particular NAT-based screening deployment being performed even after the sample has been prepared and nucleic acids isolated for amplification. Such flexibility leads to further increases in throughput and reduces liquid and solid waste by eliminating inefficient degradation of pooled samples. Such flexibility also provides the ability to perform "stat" NAT-based screening deployments, which allow samples to be processed outside of the batch order typically required with current systems.

[0095] The increased throughput and flexibility of the disclosed methods and systems can increase access to donor blood and blood products by reducing the time required to process blood, and allow donors to receive more timely information regarding the presence of pathogens or infectious agents.

[0096] To achieve the aforementioned improvements, the present disclosure is directed, in various embodiments, to innovative methods and systems for rapid, sensitive, and high-throughput NAT-based screening of donor blood (e.g., whole blood, lysed whole blood, serum, or plasma) samples that incorporate sample preparation, amplification, or detection aspects, or combinations of such aspects, as disclosed herein.

[0097] Reference will now be made in detail to various exemplary embodiments of the disclosed subject matter, which are illustrated in the accompanying drawings and described in more detail below. The methods of the disclosed subject matter will be described in conjunction with a detailed description of the system. The accompanying drawings, in which like reference numbers refer to identical or functionally similar elements throughout the different views, further illustrate various embodiments in accordance with the disclosed subject matter and serve to explain various principles and advantages.

[0098] By way of introduction, FIG. 1 generally illustrates the general steps of NAT-based screening of the disclosed subject matter. In particular, by way of example and not limitation, FIG. 1 generally illustrates the basis of methods and systems for automated NAT-based screening with improved speed and throughput. In accordance with the disclosed subject matter, by way of example and not limitation, a system for NAT-based screening is provided, along with the disclosed steps. The system includes a sample preparation component configured to prepare a sample for nucleic acid analysis, an amplification component configured to amplify one or more targets (e.g., target nucleic acids) in the sample, and a detection component configured to detect the presence or absence of one or more targets (e.g., target nucleic acids) in the sample. In certain embodiments, detection is performed after amplification is complete. Additionally or alternatively, methods and systems embodied by the general steps illustrated in FIG. 1 and described in detail herein may include combined amplification and detection aspects. For example, and without limitation, detection can be repeated or even continuous throughout the course of amplification, as embodied by the dashed arrow in Figure 1 leading from detection back to amplification (the return arrow is shown dashed to convey optionality). Each of these steps and corresponding components of the disclosed subject matter has various aspects, which will be described in further detail with respect to the advantages of the disclosed subject matter. Furthermore, as described below, it will be recognized that various aspects of each step and corresponding component can be selectively combined to achieve the desired advantages of the methods and systems disclosed herein.

[0099] With this general understanding, various unique aspects of one or more steps can be incorporated to achieve the desired benefits of the disclosed subject matter, as described in more detail below. For example, without limitation, according to one aspect of the disclosed subject matter, methods and systems for screening a sample of donor blood can perform nucleic acid analysis on the sample to detect one or more pathogens or infectious agents. In certain embodiments, a determination of a predetermined level of nucleic acid derived from each pathogen or infectious agent can be based on the performed nucleic acid analysis. This determination can indicate whether the donor blood or donor material can be shipped for clinical use or further processing. For example, the methods and systems can determine whether the donor blood is acceptable for transfusion based in part on the nucleic acid analysis results. In certain embodiments, the methods and systems can determine whether material derived from the donor is acceptable for transfusion, transplantation, or therapeutic drug manufacturing based in part on the nucleic acid analysis results. In certain embodiments, detection of a level of a pathogen or infectious agent at or above a predetermined level indicates that the material derived from the donor is unacceptable for transfusion, transplantation, or therapeutic drug manufacturing. In certain embodiments, the presence of a pathogen or infectious agent indicates that the material derived from the donor is unacceptable for transfusion, transplantation, or therapeutic manufacture. In certain embodiments, detection of a level of a pathogen or infectious agent below a predetermined level indicates that the material derived from the donor is acceptable for transfusion, transplantation, or therapeutic manufacture. In certain embodiments, the absence of a pathogen or infectious agent indicates that the material derived from the donor is acceptable for transfusion, transplantation, or therapeutic manufacture. Release of donor blood or donor material for clinical use can occur in about 15 to about 60 minutes, e.g., about 20 to about 60 minutes, about 20 to about 45 minutes, about 15 to about 45 minutes, about 20 to about 40 minutes, or about 15 to about 40 minutes, from the initial aspiration of the sample, e.g., from a sample container in a sample loading area or from a sample container in a sample tube rack in an aspiration position, to perform nucleic acid analysis in accordance with the disclosed subject matter.

[0100] Additionally or alternatively, according to another aspect of the disclosed subject matter, methods and systems for screening a sample of donor blood can include performing a nucleic acid analysis on the sample to detect one or more pathogens or infectious agents. In certain embodiments, the methods and systems can further include determining a predetermined level of nucleic acid derived from each of the pathogens or infectious agents based on the performed nucleic acid analysis. In certain embodiments, the methods and systems can further include determining the presence or absence of nucleic acid derived from each of the pathogens or infectious agents based on the performed nucleic acid analysis. In certain embodiments, the nucleic acid analysis includes a nucleic acid amplification reaction according to the disclosed subject matter, having a duration of about 1 minute to about 20 minutes, e.g., about 5 minutes to about 20 minutes or about 8 minutes to about 20 minutes. In certain embodiments, the amplification reaction includes a detection process, e.g., referred to herein as an amplification and detection process. This determination can indicate whether the donor blood or donor material can be released for clinical use. Furthermore, the methods and systems can determine whether the donor blood is acceptable for transfusion based in part on the nucleic acid analysis results. In certain embodiments, the methods and systems can determine whether a material derived from a donor is acceptable for transfusion, transplantation, or therapeutic drug manufacturing based in part on the nucleic acid analysis results. In certain embodiments, detection of a level of a pathogen or infectious agent at or above a predetermined level indicates that the material derived from the donor is unacceptable for transfusion, transplantation, or therapeutic drug manufacturing. In certain embodiments, the presence of a pathogen or infectious agent indicates that the material derived from the donor or donor blood is unacceptable for transfusion, transplantation, or therapeutic drug manufacturing. In certain embodiments, detection of a level of a pathogen or infectious agent below a predetermined level indicates that the material derived from the donor or donor blood is acceptable for transfusion, transplantation, or therapeutic drug manufacturing. In certain embodiments, the absence of a pathogen or infectious agent indicates that the material derived from the donor or donor blood is acceptable for transfusion, transplantation, or therapeutic drug manufacturing.

[0101] Additionally or alternatively, according to another aspect of the disclosed subject matter, methods and systems for screening samples of donor blood can perform nucleic acid analysis on the sample to detect one or more pathogens or infectious agents, wherein a determination of a predetermined level of nucleic acid from each of the pathogens or infectious agents can be based on the performed nucleic acid analysis, and wherein each determination of a predetermined level of nucleic acid from one of the pathogens or infectious agents can be completed in about 15 to about 45 minutes, e.g., about 20 to about 45 minutes, about 20 to about 40 minutes, or about 15 to about 40 minutes, from the initial drawing of the sample for performing a nucleic acid analysis according to the disclosed subject matter. This determination can indicate whether the donor blood can be released for clinical use. Furthermore, the methods and systems can determine whether the donor blood is acceptable for transfusion based in part on the nucleic acid analysis results. In certain embodiments, the methods and systems can determine whether material from the donor who provided the sample of donor blood can be released for clinical use based in part on the nucleic acid analysis results.

[0102] Additionally or alternatively, according to another aspect of the disclosed subject matter, methods and systems for screening a sample of donor blood can include performing nucleic acid analyses on the sample to detect one or more pathogens or infectious agents. In certain embodiments, the methods and systems can include determining a predetermined level of nucleic acid from each of the pathogens or infectious agents based on the nucleic acid analyses performed. In certain embodiments, the methods and systems can determine the presence or absence of nucleic acid from each of the pathogens or infectious agents based on the nucleic acid analyses performed. In certain embodiments, the time to obtain results for each determination of a predetermined level of nucleic acid from one of the pathogens or infectious agents is about 15 to about 45 minutes, e.g., about 20 to about 45 minutes, about 20 to about 40 minutes, or about 15 to about 40 minutes. This determination can indicate whether the sample of donor blood or the donor material can be shipped for clinical use. Furthermore, the methods and systems can determine whether the donor blood is acceptable for transfusion based in part on the nucleic acid analysis results.

[0103] Additionally or alternatively, according to another aspect of the disclosed subject matter, methods and systems for screening samples of donor blood can perform nucleic acid analysis on multiple samples to detect one or more pathogens or infectious agents. The determination of a predetermined level of nucleic acid derived from each of the pathogens or infectious agents in each of the multiple samples can be based on the performed nucleic acid analyses. In certain embodiments, the methods and systems can include determining the presence or absence of nucleic acid derived from each of the pathogens or infectious agents based on the performed nucleic acid analyses. The nucleic acid analysis-based determination is performed within about 15 minutes to about 3.5 hours, e.g., or within about 15 minutes to about 3.5 hours, or within about 20 minutes to about 3.5 hours, from the initial aspiration of the first sample from a sample preparation area for performing a nucleic acid analysis according to the disclosed subject matter. This determination can indicate whether the donor blood can be shipped for clinical use or whether the donor material can be shipped for clinical use. Furthermore, the methods and systems can determine whether the donor blood is acceptable for transfusion based in part on the nucleic acid analysis results. In certain embodiments, the methods and systems can determine whether a material derived from a donor can be shipped for clinical use based in part on the nucleic acid analysis results. For example, without limitation, the detection of the presence of a pathogen or infectious agent indicates that the donor or material derived from the donor's blood is unacceptable and cannot be shipped for clinical use. In certain embodiments, the absence of a pathogen or infectious agent indicates that the donor or material derived from the donor's blood can be shipped for clinical use.

[0104] Additionally or alternatively, according to another aspect of the disclosed subject matter, methods and systems for screening samples of donor blood can perform nucleic acid analysis on multiple samples to detect one or more pathogens or infectious agents. In certain embodiments, the determination of a predetermined level of nucleic acid from each of the pathogens or infectious agents can be based on the nucleic acid analysis performed. In certain embodiments, methods and systems perform screening to detect a volume of 1 m occupied by an automated system (e.g., used to perform the method) according to the disclosed subject matter. 3 at least about 70 results per hour per m footprint of an automated system (e.g., used to perform the method); 2 The system can generate at least about 140 results per hour per day. This determination can indicate whether the donor blood can be shipped for clinical use or whether the donor material can be shipped for clinical use. Furthermore, the method and system can determine whether the donor blood is acceptable for transfusion based in part on the nucleic acid analysis results. In certain embodiments, the method and system can determine whether material derived from the donor is acceptable for transplantation or therapeutic drug manufacturing based in part on the nucleic acid analysis results.

[0105] Additionally or alternatively, according to another aspect of the disclosed subject matter, the screening method and system can be used with pooled donor blood samples to perform nucleic acid analysis on the pooled sample to detect one or more pathogens or infectious agents. The determination of a predetermined level of nucleic acid derived from each of the pathogens or infectious agents can be based on the performed nucleic acid analysis. If it is determined that nucleic acid derived from at least one of the pathogens or infectious agents is present in the pooled sample, for example, at or above a predetermined level, the method and system can screen individual donor blood samples or subpools of donor blood included in the pooled sample to detect one or more pathogens or infectious agents by nucleic acid analysis of the samples. The determination of a predetermined level of nucleic acid derived from each of the pathogens or infectious agents can be based on the performed nucleic acid analysis. This determination can indicate whether the donor blood or donor material can be shipped for clinical use. Furthermore, the method and system can determine whether the donor blood is acceptable for transfusion based in part on the nucleic acid analysis results. In certain embodiments, the methods and systems can determine whether material from a donor is acceptable for transplantation or therapeutic drug manufacture based in part on the nucleic acid analysis results.

[0106] Additionally or alternatively, according to another aspect of the disclosed subject matter, methods and systems for screening samples of donor blood can include a sample analysis station and a memory containing a processor and instructions to be executed. The sample analysis station can include a sample loading area, a sample preparation area, a nucleic acid amplification area, and a nucleic acid detection area. When the processor executes the instructions, the system can perform nucleic acid analysis on the sample of donor blood to detect one or more pathogens or infectious agents based on the nucleic acid analysis. In certain embodiments, if a predetermined level of nucleic acid derived from each of a plurality of pathogens or infectious agents is determined based on the nucleic acid analysis, the method and system indicates that the donor blood is ready for shipment for clinical use or that the donor material can be shipped for clinical use. In certain embodiments, if a predetermined level of nucleic acid derived from each of a plurality of pathogens or infectious agents is determined based on the nucleic acid analysis, the method and system indicates that the donor blood or donor material can be shipped for clinical use. Furthermore, the method and system can determine whether the donor blood is acceptable for transfusion based in part on the nucleic acid analysis results. As embodied herein, screening and nucleic acid analysis of multiple donor blood samples for shipment of donor blood or donor material for clinical use is performed independently, i.e., without the need to screen multiple samples in a predetermined order or to predetermine a particular nucleic acid analysis prior to contact with sample lysis buffer. Additionally or alternatively, such independent nucleic acid analysis allows for separate analyses to be performed without impacting sample throughput or time to results (TTR), regardless of whether the sample precedes or follows a particular sample.

[0107] Before describing certain aspects of the methods and systems of the present disclosure in detail below, it is desirable to first describe exemplary methods and systems used herein. As embodied herein, the exemplary methods can be performed on a system including a sample analysis station. By way of example and not limitation, the sample analysis station can include a sample loading area, a sample preparation area, a nucleic acid amplification area, and a nucleic acid detection area. The exemplary methods can include performing a sample preparation process in the sample preparation area. Additionally or alternatively, the exemplary methods can include performing an amplification process in the amplification area. Additionally or alternatively, the exemplary methods can include performing a detection process in the detection area. By way of example and not limitation, the exemplary methods can include performing a sample preparation process in the sample preparation area, performing an amplification process in the amplification area, and performing a detection process in the detection area.

[0108] By way of example and not limitation, the sample preparation area can include a sample transport system and a washing and elution system. For example, as embodied herein, the sample preparation area can include a sample transport configured to transport one or more samples in containers from a sample dispensing position to a sample capture and transfer position along a transport path. For example, as embodied herein, the sample transport can include a sample preparation carousel, e.g., a lysis carousel. For example, as embodied herein, an exemplary sample preparation process can include dispensing a sample into a container at a sample dispensing position of the sample transport. For example, but not by way of limitation, the sample can be dispensed using a pipettor. The exemplary sample preparation process can further include transporting the sample in the container along a transport path of the sample transport to the sample capture and transfer position. By way of example and not limitation, as described further herein, the exemplary sample preparation process can include performing a lysis process, a pretreatment process, and / or an on-board pooling process on the sample transport of the sample preparation area. As described further herein, the exemplary pretreatment and on-board pooling processes can include transferring samples between containers on the sample transport as the containers are transported along the transport path. For example, as embodied herein, the sample preparation area can include a pipettor that can transfer, e.g., aspirate and dispense, a sample from a container on the sample transporter into a container on the sample transporter.

[0109] An exemplary sample preparation process can further include transferring the sample from the sample transporter to a washing and elution system. For example, as embodied herein, the sample preparation area can include a particle transfer mechanism, which can transfer the particles and their bound nucleic acids, e.g., CuTi-coated microparticles, to the washing and elution system. As further described herein, an exemplary sample preparation process can include a washing process. An exemplary washing process can include one or more washing steps to wash the nucleic acid-bound microparticles, e.g., CuTi-coated microparticles bound to the nucleic acids. Additionally or alternatively, an exemplary washing process can include an elution step. For example, as embodied herein, a washing process can include three washing steps and one elution step.

[0110] Additionally or alternatively, exemplary methods can include performing an amplification process in an amplification region and performing a detection process in a detection region. For illustrative purposes, and as embodied herein, the amplification region and the detection region can include an amplification and detection system, and the amplification process and the detection process can include an amplification and detection process. The amplification and detection system can include, for example, a carousel having one or more amplification vessels and one or more detectors. As embodied herein, a sample can be transferred from a sample preparation region to the amplification and detection region for the amplification and detection process. By way of example and not limitation, eluate from a wash process can be transferred to the amplification and detection system, for example, using a pipettor. Exemplary amplification and detection methods can include amplifying a target nucleic acid and simultaneously detecting the resulting amplicons, for example, as further described herein.

[0111] In certain embodiments, exemplary methods include performing a nucleic acid analysis on a sample of donor blood, the nucleic acid analysis including a sample preparation process and an amplification and detection process. In certain embodiments, the nucleic acid analysis is completed in about 15 minutes to about 34 minutes, wherein the sample preparation process is completed in about 14 minutes and the amplification and detection process is completed in about 1 to about 20 minutes.

[0112] In certain embodiments, the sample preparation process includes providing a sample of donor blood, e.g., a whole blood sample, a plasma sample, or a serum sample, e.g., from a sample loading area, as shown at 7901 in FIG. 79 . In certain embodiments, the exemplary method may further include a sample preparation process. For example, as shown at 7902 in FIG. 79 , the sample preparation process may include a lysis process, e.g., lysing the sample in a lysis vessel. The lysis process may be performed on a sample transporter, e.g., a sample preparation carousel, such as lysis carousel 6805 or 8001. In certain embodiments, the lysis process may include combining the sample with a lysis buffer, microparticles, e.g., CuTi-coated microparticles, and proteinase K to generate a mixture. In certain embodiments, the lysis process and lysis of the sample do not require a separate proteinase K treatment step. In certain embodiments, the lysis process, e.g., lysing the sample, may further include combining the mixture with an internal control nucleic acid or calibrator. In certain embodiments, the exemplary method may further include incubating the mixture to promote binding of nucleic acids in the mixture to the microparticles. As shown in Figure 4, CuTi microparticles can bind to both RNA and DNA in samples containing target nucleic acids (eg, pathogenic nucleic acids) and non-target nucleic acids (eg, host nucleic acids).

[0113] In certain embodiments, the sample preparation process of the exemplary method can include a washing process. In certain embodiments, the washing process can include washing the nucleic acid-bound microparticles in a first wash. For example, the washing process can include a first wash, as shown at 7903 in FIG. 79. In certain embodiments, the first wash can include a lysis buffer. In certain embodiments, the nucleic acid-bound microparticles can be transferred from a sample transporter, such as a lysis vessel, to another vessel for the first wash. For example, the nucleic acid-bound microparticles can be transferred from the sample transporter to a washing and elution system. For example, without limitation, the microparticles can be transferred using particle transfer mechanism 6803 from a lysis vessel in a sample transporter, such as lysis carousel 6805 or 8001, to a washing vessel in a washing and elution system, such as wash track 6801 or 8002. In certain embodiments, a washing vessel for use in the present disclosure has the structure shown in FIGS. 42, 71, and 79. For example, without limitation, a washing vessel for use in the present disclosure can include two or more wells, such as four wells. As embodied herein, the wash and elution system can include a wash track, e.g., wash track 6801 or 8002. The wash track of the disclosed system can be configured to perform the wash steps of the disclosed methods and can further include multiple wash vessels. By way of example and not limitation, the wash track, e.g., wash track 6801 or 8002, can be in the shape of a racetrack. In certain embodiments, the wash process of the exemplary method can subsequently include washing the nucleic acid-bound microparticles twice with water, e.g., a second wash with water and a third wash with water, as shown, e.g., at 7904 in FIG. 79 . In certain embodiments, the second and third washes are performed in different wells from the first wash, e.g., by applying a magnetic force to capture the microparticles on the inner surface of a first wall of the first well and then moving the captured microparticles along the inner surface of the first wall to the second / third well.For purposes of illustration and not limitation, particles can move within or across a well in about 24 seconds, as shown in Figure 78. In certain embodiments, nucleic acids bound to the microparticles can be eluted (e.g., using an elution buffer and / or by heat) to produce an eluate in a fourth well of the wash vessel, for example, as shown at 7905 in Figure 79.

[0114] In certain embodiments, the exemplary method further includes performing an amplification and detection process. In certain embodiments, amplification and detection are performed simultaneously. In certain embodiments, the amplification and detection process of the exemplary method includes preparing an eluate for an isothermal amplification reaction to amplify a target nucleic acid of interest in the eluate, as shown, for example, at 7906 in FIG. 79 , and simultaneously detecting the amplified target nucleic acid. As embodied herein, after elution, the target nucleic acid can be transferred to an amplification and detection system 6807. By way of example and not limitation, the amplification and detection system can include a carousel. For example, amplification and detection system 6807 includes carousel 8003. In certain embodiments, preparing the eluate for amplification and detection includes contacting the eluate with a reagent mixture and an activating agent, such as magnesium. In certain embodiments, the reagent mixture, as referred to herein, can be an "RPA master mix," as shown, for example, in FIG. 79 . In certain embodiments, the RPA master mix includes seven enzymes that facilitate primer binding and extension in the amplification reaction, such as (i) a recombinase (e.g., UvsX), (ii) a single-stranded binding protein (e.g., GP32), (iii) a recombinase loading agent (e.g., UvsY), (iv) a DNA polymerase, (v) an exonuclease (e.g., exonuclease III), (vi) a creatine kinase, and (vii) a reverse transcriptase (e.g., EIAV-RT). In certain embodiments, for example, when the target nucleic acid is DNA, the RPA master mix does not include a reverse transcriptase. In certain embodiments, the RPA master mix can further include one or more primers that bind to the target nucleic acid and one or more probes that bind to the amplicon and facilitate signal generation. In certain embodiments, the RPA master mix can further include one or more non-protein components (e.g., for extending primers (e.g., dNTPs), for use as an energy source (e.g., ATP and phosphocreatine), for stabilizing proteins and the reaction (e.g., reaction buffer (e.g., Tris and salt)), and for use as a crowding agent (e.g., polyethylene glycol)).

[0115] In certain embodiments, an exemplary method can include amplifying a target nucleic acid of interest using an isothermal amplification reaction and simultaneously detecting the resulting amplicon, e.g., by fluorescent detection. For example, without limitation, an exemplary method can include amplifying a target nucleic acid of interest using an isothermal amplification reaction and simultaneously detecting the resulting amplicon, e.g., by fluorescent detection, as shown at 7907 in FIG. 79. Non-limiting examples of isothermal amplification reactions can include transcription-mediated amplification (TMA), recombinase polymerase amplification (RPA), and nicking enzyme amplification reaction (NEAR). In certain embodiments, the isothermal amplification reaction is RPA. The use of isothermal amplification eliminates the need for time-consuming temperature transitions. As embodied herein, an amplification and detection system can include independent fluorescent detectors, e.g., about five independent fluorescent detectors, for detecting fluorescent signals at predetermined intervals, e.g., about every 24 seconds, during the amplification reaction. For example, without limitation, an amplification and detection system 6807 can include about five independent fluorescent detectors for detecting fluorescent signals at predetermined intervals, e.g., about every 24 seconds, as shown in FIG. 78. In certain embodiments, exemplary methods may further include determining results from the nucleic acid analysis, e.g., determining the presence of nucleic acid from at least one of the plurality of pathogens or infectious agents at or above a predetermined level in the sample of donor blood, determining the presence of nucleic acid from at least one of the plurality of pathogens or infectious agents in an amount below a predetermined level in the sample of donor blood, determining the presence of nucleic acid from at least one of the plurality of pathogens or infectious agents in the sample of donor blood, or determining the absence of nucleic acid from at least one of the plurality of pathogens or infectious agents in the sample of donor blood.

[0116] In certain embodiments, exemplary methods and systems of the present disclosure can be used to perform nucleic acid analysis on a sample. In certain embodiments, exemplary methods and systems of the present disclosure can be used to screen individual or pooled blood donors (e.g., whole blood, lysed whole blood, serum, or plasma), for example, to determine whether a donor sample is acceptable for transfusion, and to screen organ and / or tissue donors to determine whether material derived from the donor (i.e., donor material) is acceptable for clinical use. In certain embodiments, exemplary methods and systems of the present disclosure can be used for quantitative and / or qualitative detection of nucleic acids derived from pathogens or infectious agents in a sample, e.g., a sample of donor blood. Additional examples of samples analyzed using the disclosed methods and systems are described herein. In certain embodiments, exemplary methods and systems of the present disclosure can be used for qualitative detection of nucleic acids derived from pathogens or infectious agents in a sample, e.g., a sample of donor blood. For example, without limitation, exemplary methods and systems of the present disclosure can be used to determine the presence or absence of nucleic acids derived from pathogens or infectious agents in a sample, e.g., a sample of donor blood. In certain embodiments, exemplary methods and systems of the present disclosure can be used for quantitative detection of nucleic acids derived from pathogens or infectious agents in a sample, such as a sample of donor blood. In certain embodiments, exemplary methods and systems of the present disclosure can be used to detect and / or quantify nucleic acids derived from pathogens, including, but not limited to, HIV-1, HIV-2, HCV, HBV, WNV, Zika virus, Chikungunya virus, Dengue virus, Babesia, malaria (e.g., by detecting and / or quantifying Plasmodium, which causes malaria), Parvovirus B19, HAV, and / or HEV. In certain embodiments, exemplary methods and systems of the present disclosure can be used for qualitative detection of nucleic acids derived from one or more pathogens or infectious agents.In certain embodiments, exemplary methods and systems of the present disclosure can be used for the qualitative detection of nucleic acids derived from HIV-1, HIV-2, HBV, HCV, parvovirus B19, HAV, WNV, Zika virus, dengue virus, chikungunya virus, babesia, malaria, Ustu virus, and / or HEV. In certain embodiments, exemplary methods and systems of the present disclosure can be used for the qualitative detection of nucleic acids derived from SARS-CoV-2 (COVID-19), coronavirus, HIV-1, HIV-2, HBV, HCV, CMV, Epstein-Barr virus (EBV), human T-lymphotropic virus (HTLV), parvovirus B19, HAV, syphilis, chlamydia, gonorrhea, dengue, chikungunya, WNV, HEV, Ustu virus, and / or Creutzfeldt-Jakob disease (vCJD). In certain embodiments, exemplary methods and systems of the present disclosure can be used for the qualitative detection of nucleic acids derived from HIV-1, HIV-2, HCV, HBV, and WNV in serum or plasma samples. In certain embodiments, exemplary methods and systems of the present disclosure can be used for multiplex analysis of HIV-1, HIV-2, HCV, and HBV in serum or plasma samples. In certain embodiments, exemplary methods and systems of the present disclosure can be used for the qualitative detection of nucleic acids derived from Babesia in whole blood samples. In certain embodiments, exemplary methods and systems of the present disclosure can be used for the qualitative detection of nucleic acids derived from HAV in plasma samples. In certain embodiments, exemplary methods and systems of the present disclosure can be used for the quantitative detection of nucleic acids derived from parvovirus B19 in plasma samples.

[0117] In certain embodiments, the exemplary methods and systems of the present disclosure can be used for the quantitative detection of nucleic acids derived from one or more pathogens or infectious agents disclosed herein. For example, but not by way of limitation, the exemplary methods and systems of the present disclosure can be used for the quantitative detection of nucleic acids derived from parvovirus B19.

[0118] In certain embodiments, exemplary methods of the present disclosure can be completed using a system disclosed herein in about 15 minutes to about 60 minutes, e.g., about 15 minutes to about 60 minutes from the initial aspirating of a sample for lysis from a sample container, e.g., a sample container present in the sample loading area or a sample container in a sample tube rack in the aspiration position. In certain embodiments, exemplary methods of the present disclosure can be completed using a system disclosed herein in about 15 minutes to about 45 minutes, about 15 minutes to about 40 minutes, or about 15 minutes to about 34 minutes, e.g., about 15 minutes to about 45 minutes from the initial aspirating of a sample for lysis from a sample container in a sample tube rack in the aspiration position or from the sample loading area. In certain embodiments, exemplary methods of the present disclosure can be completed using a system disclosed herein in about 20 minutes to about 60 minutes, e.g., about 20 minutes to about 60 minutes from the initial aspirating of a sample for lysis. In certain embodiments, exemplary methods of the present disclosure can be completed using the systems disclosed herein in about 20 to about 45 minutes, e.g., from the initial aspiration of a sample for lysis from a sample container, e.g., a sample container in a sample tube rack in the aspiration position, or a sample container present in a sample loading area, or from about 20 to about 45 minutes, or from about 20 to about 34 minutes. For example, and without limitation, exemplary methods of the present disclosure can be completed using the systems disclosed herein in about 35 minutes or less from the initial aspiration of a sample for lysis to produce a result, e.g., determining the amount of target nucleic acid amplified in an amplification reaction.

[0119] In certain embodiments, nucleic acid analysis begins with aspiration of a sample from a sample container, for example, in a sample loading area or in a sample tube rack in the aspiration position, and ends with determination of the results. In certain embodiments, nucleic acid analysis begins with aspiration of a sample from a sample container, for example, in a sample loading area or in a sample tube rack in the aspiration position, and ends at the end of incubation of the sample in an amplification container on the amplification and detection system. In certain embodiments, nucleic acid analysis can be completed in about 15 to about 36 minutes, about 16 to about 36 minutes, about 17 to about 36 minutes, about 18 to about 36 minutes, about 19 to about 36 minutes, about 20 to about 30 minutes, about 33 to about 35 minutes, or about 32 to about 36 minutes. In certain embodiments, nucleic acid analysis can be completed in about 15 minutes, about 16 minutes, about 17 minutes, about 18 minutes, about 19 minutes, about 20 minutes, about 21 minutes, about 22 minutes, about 23 minutes, about 24 minutes, about 25 minutes, about 26 minutes, about 27 minutes, about 28 minutes, about 29 minutes, about 30 minutes, about 31 minutes, about 32 minutes, about 33 minutes, about 34 minutes, about 35 minutes, about 36 minutes, about 37 minutes, about 38 minutes, about 39 minutes, about 40 minutes, about 41 minutes, about 42 minutes, about 43 minutes, about 44 minutes, or about 45 minutes. In certain embodiments, nucleic acid analysis can be completed in about 15 minutes. In certain embodiments, nucleic acid analysis can be completed in about 34 minutes. In certain embodiments, exemplary methods and systems of the present disclosure can be used to obtain at least about 150 to about 300 results per hour. In certain embodiments, exemplary methods and systems of the present disclosure can be used to obtain at least about 1,000 to about 2,500 results per 8 hours, for example, about 1,100 to about 2,300 results per 8 hours. In certain embodiments, exemplary methods and systems of the present disclosure can be used to obtain at least about 1,000 to about 2,500 results per 8 hours. 2 At least about 500 to about 1,200 results per 8 hours, e.g., for an automated system with a footprint of 1 m 2 At least about 570 to about 1,150 results can be obtained per 8 hours.

[0120] For purposes of clarity, and not by way of limitation, the detailed description of the subject matter disclosed herein is divided into the following subsections: 1. Mode of sample collection; 2. Aspects of sample preparation; 3. Aspects of nucleic acid amplification; 4. Aspects of nucleic acid detection; 5. How to use; 6. Analysis system; 7. Additional screening and preparative aspects; and 8. Exemplary Embodiments.

[0121] The terms used herein generally have their ordinary meanings in the art, within the context of this disclosure and in the specific context in which each term is used. Certain terms are discussed below or elsewhere in the specification to provide additional guidance to the practitioner in describing the compositions and methods of the present disclosure, and how to make and use them.

[0122] As used herein, the term "donor" refers to any animal (e.g., mammal), including but not limited to, a human, a non-human primate, a rodent, etc., that provides a biological sample. For example, but not limited to, the biological sample can be blood, serum, or plasma, e.g., used in a transfusion.

[0123] As used herein, "donor blood" refers to blood obtained from a donor, e.g., whole blood, lysed whole blood, serum, or plasma, as well as products derived from such blood, e.g., platelets, packed red blood cells, and products derived from plasma, including, but not limited to, (1) clotting factors, e.g., factor VIII, von Willebrand factor, fibrinogen; (2) protease inhibitors, e.g., α1-antitrypsin and C1-esterase inhibitor; (3) albumin; and (4) immunoglobulin G (IgG).

[0124] As used herein, the term "patient" refers to any animal (e.g., mammal), including but not limited to humans, non-human primates, and rodents, that is to receive a particular clinical treatment, e.g., a blood transfusion.

[0125] As used herein, the phrase "clinical use" refers to "in vivo clinical use" and "in vitro clinical use." As used herein, "in vivo clinical use" refers to the transfusion of whole blood as well as the transfusion of components of whole blood, collectively referred to herein as "blood products," e.g., packed red blood cells, plasma (e.g., unprocessed frozen plasma or thawed plasma), platelets, or cryoprecipitate (prepared by thawing unprocessed frozen plasma and collecting the precipitate). "In vivo clinical use" also encompasses the incorporation of donor blood, or materials derived therefrom, in the manufacture of therapeutics, as well as the donation and / or transplantation of one or more materials, e.g., organs, tissues, etc., from a donor. For example, without limitation, donor blood can be processed into plasma, either after collection as whole blood or as a direct plasma donation via automated apheresis methods in which blood is removed from the donor, the plasma is collected, and the remaining blood is returned to the donor. The plasma can be used directly as unprocessed frozen plasma or further processed to produce a variety of therapeutic biologics known as plasma-derived products. For example, but not limited to, plasma can be pooled to a significant extent, e.g., pools of 10,000 to 50,000 donations can be combined for industrial processing, and the pooled plasma can be fractionated to produce plasma-derived products, including, but not limited to, (1) clotting factors, e.g., factor VIII, von Willebrand factor, and fibrinogen; (2) protease inhibitors, e.g., α1-antitrypsin and C1-esterase inhibitor; (3) albumin; and (4) immunoglobulin G (IgG). As used herein, "in vitro clinical use" refers to the use of blood products, plasma-derived products, or biological materials other than direct transfusion or transplantation of donor material into patients, e.g., in research and development of new medical devices, therapeutic processes, or disease diagnosis, as well as in connection with quality assurance / laboratory diagnostics.

[0126] As used herein, the phrase "donor material" refers to blood products and other biological products, including, for example, tissues, organs, vaccines, cells, gene therapies, and recombinant therapeutic proteins. Donor material can include multiple donor materials. For example, donor material can include multiple blood products and / or other biological products from a single donor. Additionally or alternatively, donor material can include blood products from multiple donors, biological products from multiple donors, or blood products from one donor and other biological products from one or more other donors.

[0127] As used herein, the phrase time to result (“TTR”) refers to the time from the initiation of a nucleic acid analysis, including, for example, sample preparation, amplification, and detection, to the completion of the detection step of the nucleic acid analysis. In certain embodiments, for performing a nucleic acid analysis in accordance with the disclosed subject matter, the initiation of the nucleic acid analysis occurs upon initial aspiration of a sample, for example, from a sample container in the sample loading area or from a sample container in a sample tube rack in the aspiration position. In certain embodiments, the sample container comprises a container in the sample loading area. For example, in certain embodiments, aspiration of a sample for a nucleic acid analysis can occur at an aspiration position, e.g., aspiration position 6292. Additionally or alternatively, aspiration of a sample for a nucleic acid analysis can occur in an inner portion of a sample loading area, e.g., sample loading area 3102 in FIG. 54B . In certain embodiments, TTR refers to the time from the initiation of a nucleic acid analysis, for example, from the initial aspiration of a sample from the sample loading area or from a sample container in a sample tube rack in the aspiration position, to the completion of a result during the amplification and detection process. In certain embodiments, TTR refers to the time from the start of a nucleic acid analysis, for example, from the initial aspiration of a sample from a sample loading area or from a sample container in a sample tube rack at an aspiration position, to the completion of the amplification and detection process.

[0128] As used herein, "result" refers to the detection of the presence of one or more target nucleic acids. In certain embodiments, results obtained using the disclosed methods and systems can include determining the absence of one or more target nucleic acids. In certain embodiments, results obtained using the disclosed methods and systems can include the detection of one or more target nucleic acids at or above a predetermined level. In certain embodiments, results obtained using the disclosed methods and systems can include the detection of one or more target nucleic acids at amounts less than a predetermined level. In certain embodiments, results obtained using the disclosed methods and systems can include quantification of one or more target nucleic acids. In certain embodiments, as embodied in Figures 67A and 67B, a single sample aspiration can result in an eluate (e.g., referred to herein as "dispensed eluate") that is dispensed into two amplification reactions, thereby generating two or more "results." As used herein, a result can include, for example, the detection of the presence or absence of one or more target nucleic acids. For example, and without limitation, scenarios 2 and 4 in Figure 67B each illustrate the use of a single sample preparation process followed by two amplification reactions, which are multiplexed to detect two target nucleic acids, each from a different pathogen or infectious agent. Each of these scenarios illustrates the generation of four results, as each amplification reaction provides information about two target nucleic acids, making it possible to detect the presence or absence of four pathogens or infectious agents in a single sample using two amplification reactions. Additionally or alternatively, higher-order multiplex amplifications can be utilized in conjunction with the disclosed methods and systems, thereby detecting the presence of 5, 6, 7, 8, 9, 10, or more pathogens or infectious agents in a single sample, thereby providing 5, 6, 7, 8, 9, 10, or more results, respectively. In certain embodiments, each result includes a distinct detection of the presence, absence, or level of a target nucleic acid.

[0129] As used herein, TTR refers to the time from the start of nucleic acid analysis, and therefore necessarily excludes the time that occurs between donation and its screening by the HTNAT analysis system or method described herein. For example, but not limited to, the time spent on sampling, such as collecting donor blood, or sample transport time, such as transporting the sample from a local blood collection center or plasma center to a central laboratory for screening, is excluded from TTR.

[0130] As used herein, the term "throughput" refers to the number of nucleic acid analysis results obtained per unit time, e.g., per hour. Additionally or alternatively, throughput can refer to, but is not limited to, the number of samples analyzed per unit time, e.g., per hour. In certain embodiments, throughput can refer to, but is not limited to, the number of tests performed per unit time, e.g., per hour.

[0131] As used herein, the use of the words "a" or "an," when used in conjunction with the word "comprising" in the claims and / or specification, may mean "one," but is also consistent with the meanings of "one or more," "at least one," and "one or more than one."

[0132] For example, the term "certain" as used in connection with "certain embodiments" and "certain aspects" refers to "exemplary" and not limiting.

[0133] As used herein, the terms "comprise(s)," "include(s)," "having," "has," "can," and "contain(s)," and variations thereof, are intended to be open-ended transitional phrases, terms, or words that do not preclude additional acts or structures. The present disclosure contemplates other embodiments that "comprising," "consisting of," and "consisting essentially of" the embodiments or elements presented herein, whether or not explicitly stated.

[0134] The term "about" or "approximately" refers to within an acceptable error range of a particular value as determined by those skilled in the art, which depends in part on how the value is measured or determined, i.e., the limitations of the measurement system. For example, "about" can mean within 3 standard deviations or more than 3 standard deviations, according to the practice in the art. Alternatively, "about" can mean a range of up to 20%, preferably up to 10%, more preferably up to 5%, and even more preferably up to 1% of a given value. Alternatively, particularly with respect to biological systems or processes, this term can mean within an order of magnitude of a value, preferably within 5-fold, more preferably within 2-fold.

[0135] The term "expression" as used herein refers to transcription and translation occurring within a cell. The expression level of a gene and / or nucleic acid within a cell can be determined based on either the amount of corresponding mRNA present in the cell or the amount of protein encoded by the gene and / or nucleic acid produced by the cell. For example, mRNA transcribed from a gene and / or nucleic acid is preferably quantified by Northern hybridization. Sambrook et al., Molecular Cloning: A Laboratory Manual, pp. 7.3-7.57 (Cold Spring Harbor Laboratory Press, 1989). Proteins encoded by a gene and / or nucleic acid can be quantified either by assaying the biological activity of the protein or by using assays independent of such activity, such as Western blotting or radioimmunoassay, using antibodies capable of reacting with the protein. Sambrook et al., Molecular Cloning: A Laboratory Manual, pp. 18.1-18.88 (Cold Spring Harbor Laboratory Press, 1989).

[0136] As used herein, the terms "nucleic acid," "nucleic acid molecule," or "polynucleotide" refer to any compound and / or substance comprising a polymer of nucleotides. Each nucleotide consists of a base, specifically a purine or pyrimidine base (i.e., cytosine (C), guanine (G), adenine (A), thymine (T), or uracil (U)), a sugar (i.e., deoxyribose or ribose), and a phosphate group. Nucleic acid molecules are often described by the sequence of bases, which represent the primary (linear) structure of the nucleic acid molecule. The sequence of bases is typically expressed from 5' to 3'. As used herein, the term nucleic acid molecule encompasses deoxyribonucleic acid (DNA), including, for example, complementary DNA (cDNA) and genomic DNA; ribonucleic acid (RNA), particularly messenger RNA (mRNA); synthetic forms of DNA or RNA; and mixed polymers containing two or more of these molecules. Nucleic acid molecules can be linear or circular. In addition, the term nucleic acid molecule includes both sense and antisense strands, as well as single- and double-stranded forms. Furthermore, the nucleic acid molecules described herein can contain naturally occurring or non-naturally occurring nucleotides. Examples of non-naturally occurring nucleotides include modified nucleotide bases having derivatized sugar or phosphate backbone linkages, or chemically modified residues.

[0137] As used herein, the term "oligonucleotide" refers to a short nucleic acid sequence containing about 2 to about 100 nucleotides (e.g., about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 99, or 100 nucleotides, or a range defined by any of the foregoing values). As used herein, the terms "nucleic acid" and "polynucleotide" refer to polymeric forms of nucleotides of any length, either ribonucleotides (RNA) or deoxyribonucleotides (DNA). These terms refer to the primary structure of the molecule and thus include double-stranded and single-stranded DNA, double-stranded and single-stranded RNA. The terms also include, as equivalents, analogs of either RNA or DNA made from nucleotide analogs and modified polynucleotides, e.g., methylated and / or capped polynucleotides. Nucleic acids are typically linked via phosphate linkages to form nucleic acid sequences or polynucleotides, although many other linkages are known in the art (eg, phosphorothioate, boranophosphate, etc.).

[0138] Oligonucleotides can be single-stranded or double-stranded, or can contain portions of both double-stranded and single-stranded sequences. Oligonucleotides can be DNA, both genomic and complementary DNA (cDNA), RNA, or hybrids, where the nucleic acid can contain combinations of deoxyribonucleotides and ribonucleotides and base combinations including uracil, adenine, thymine, cytosine, guanine, inosine, xanthine, hypoxanthine, isocytosine, and isoguanine. Oligonucleotides can be obtained by chemical synthesis or recombinant methods.

[0139] Any of the oligonucleotides described herein can be modified in any suitable manner to stabilize or enhance the binding affinity of the oligonucleotide to its target.For example, the oligonucleotide sequences described herein can include one or more modified oligonucleotide bases.

[0140] Any of the oligonucleotide sequences described herein can comprise, consist essentially of, or consist of the complement of any of the sequences disclosed herein. As used herein, the term "complement" or "complementary sequence" refers to a nucleic acid sequence that forms a stable duplex with an oligonucleotide described herein via Watson-Crick base pairing rules, and typically shares greater than about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the disclosed oligonucleotide.

[0141] The oligonucleotides described herein can be prepared using any suitable method known in the art. (e.g. Sambrook et al., Molecular Cloning. A Laboratory Manual, 1989, 2. Supp. Ed., Cold Spring Harbor Laboratory Press: New York, NY; MA Innis (Ed.), PCR Protocols. A Guide to Methods and Applications, Academic Press: New York, NY (1990); P. Tijssen, Hybridization with Nucleic Acid Probes - Laboratory Techniques in Biochemistry and Molecular Biology (Parts I and II), Elsevier Science (1993); MA Innis (Ed.), PCR Strategies, Academic Press: New York, NY (1995); and FM Ausubel (Ed.), Short Protocols in Molecular Biology, John Wiley & Sons: Secaucus, NJ (2002); Narang et al., Meth. Enzymol., 68: 90-98 (1979);Brown et al., Meth. Enzymol., 68: 109-151 (1979); and Belousov et al., Nucleic Acids Res., 25: 3440-3444 (1997), each of which is incorporated herein by reference in its entirety.) Oligonucleotide pairs can also be designed using various tools, such as the Primer-BLAST tool provided by the National Center for Biotechnology Information (NCBI).Oligonucleotide synthesis can be performed using commercially available oligo synthesizers, such as those available from Perkin Elmer / Applied Biosystems, Inc. (Foster City, CA), DuPont (Wilmington, DE), or Milligen (Bedford, MA). Alternatively, oligonucleotides can be custom made and obtained from a variety of commercial sources known in the art, including, for example, Midland Certified Reagent Company (Midland, TX), Eurofins Scientific (Louisville, KY), BioSearch Technologies, Inc. (Novato, CA), and the like. Oligonucleotides can be purified using any suitable method known in the art, such as, for example, native acrylamide gel electrophoresis, anion exchange HPLC (see, e.g., Pearson et al., J. Chrom., 255: 137-149 (1983), incorporated herein by reference), and reverse phase HPLC (see, e.g., McFarland et al., Nucleic Acids Res., 7: 1067-1080 (1979), incorporated herein by reference).

[0142] The sequence of the oligonucleotides can be verified using any suitable sequencing method known in the art, including, but not limited to, chemical degradation (see, e.g., Maxam et al., Methods of Enzymology, 65: 499-560 (1980), incorporated herein by reference), matrix-assisted laser desorption / ionization time-of-flight (MALDI-TOF) mass spectrometry (see, e.g., Pieles et al., Nucleic Acids Res., 21: 3191-3196 (1993), incorporated herein by reference), combined alkaline phosphatase digestion and exonuclease digestion followed by mass spectrometry (Wu et al., Anal. Biochem., 290: 347-352 (2001), incorporated herein by reference), and the like.

[0143] As used herein, the terms "primer," "primer sequence," "primer oligonucleotide," and "amplification oligonucleotide" refer to an oligonucleotide that can act as a point of initiation for the synthesis of an extension product, which is the complementary strand of a nucleic acid (any type of DNA or RNA), when placed under suitable amplification conditions (e.g., buffer, salt, temperature, and pH) in the presence of nucleotides and a nucleic acid polymerization agent (e.g., a DNA- or RNA-dependent polymerase). Amplification oligonucleotides of the present disclosure can be of any suitable size and desirably comprise, consist essentially of, or consist of about 15-50 nucleotides, preferably about 20-40 nucleotides. Oligonucleotides of the present disclosure can contain additional nucleotides in addition to the nucleotides described herein.

[0144] The terms "probe," "probe sequence," and "probe oligonucleotide" refer to an oligonucleotide that can selectively hybridize to at least a portion of a target sequence (e.g., a portion of an amplified target sequence) under appropriate hybridization conditions. Generally, probe sequences are identified as either "complementary" (i.e., complementary to the coding or sense strand (+)) or "reverse complementary" (i.e., complementary to the antisense strand (-)). Probes of the present disclosure can be of any suitable size and desirably comprise, consist essentially of, or consist of about 10-50 nucleotides, preferably about 12-35 nucleotides.

[0145] As used herein, the terms "set," "primer set," "probe set," and "primer and probe set" refer to two or more oligonucleotides that can together prime the amplification of a target sequence or nucleic acid of interest (e.g., a target sequence in an infectious pathogen) and / or at least one probe that can detect the target sequence or nucleic acid. In certain embodiments, the term "set" refers to a pair of oligonucleotides that includes a first oligonucleotide, referred to herein as a "forward primer," that hybridizes to the 5' end of the target sequence or nucleic acid to be amplified, and a second oligonucleotide, referred to herein as a "reverse primer," that hybridizes to the complement of the target sequence or nucleic acid to be amplified.

[0146] As used herein, the terms "target nucleic acid," "target sequence," or "target nucleic acid sequence" refer to a nucleic acid sequence, or its complement, of a pathogen or infectious agent, such as a virus, bacterium, or eukaryotic parasite, as described herein. In certain embodiments, the target sequence or target nucleic acid sequence can be detected using the methods and systems of the present disclosure.

[0147] As used herein, "sequence identity" or "identity" in the context of two polynucleotide or polypeptide sequences refers to the nucleotide bases or amino acid residues in the two sequences that are identical when aligned to obtain maximum agreement over a specified comparison window. When the percentage of sequence identity or similarity is used in relation to proteins, it is recognized that non-identical residue positions often differ by conservative amino acid substitutions, in which amino acid residues are replaced with functionally equivalent amino acid residues that have similar physicochemical properties, thus leaving the functional properties of the molecule unchanged.

[0148] As used herein, "percentage of sequence identity" or "percentage of identity" refers to a value determined by comparing two optimally aligned sequences over a comparison window, where a portion of the polynucleotide sequence within the comparison window may contain additions or deletions (gaps) compared to the reference sequence (which does not contain additions or deletions) due to optimal alignment of the two sequences. The percentage is calculated by determining the number of positions where identical nucleic acid bases or amino acid residues occur in both sequences, determining the number of matched positions, dividing the number of matched positions by the total number of positions in the comparison window, and multiplying the result by 100 to determine the percentage of sequence identity.

[0149] As will be understood by those skilled in the art, the determination of percent identity between any two sequences can be accomplished using certain well-known mathematical algorithms. Non-limiting examples of such mathematical algorithms include the Myers and Miller algorithm, the Smith et al. local homology algorithm; the Needleman and Wunsch homology alignment algorithm; the Pearson and Lipman similarity search method; and the Karlin and Altschul algorithm, modified as in Karlin and Altschul. A computer implementation of a suitable mathematical algorithm can be used to compare sequences to determine sequence identity. Such implementations include, but are not limited to, CLUSTAL, ALIGN, GAP, BESTFIT, BLAST, and FASTA, among others, which can be identified by those skilled in the art. Sequence alignment algorithms are described, for example, in Altschul et al., J. Molecular Biol., 215(3): 403-410 (1990); Beigert et al., Proc. Natl. Acad. Sci. USA, 106(10): 3770-3775 (2009), Durbin et al., eds., Biological Sequence Analysis: Probalistic Models of Proteins and Nucleic Acids, Cambridge University Press, Cambridge, UK (2009); Soding, Bioinformatics, 21(7): 951-960 (2005); Altschul et al., Nucleic Acids Res., 25(17): 3389-3402 (1997); and Gusfield, Algorithms on Strings, Trees and Sequences, Cambridge University Press, Cambridge UK. (1997), each of which is incorporated herein by reference in its entirety.

[0150] As used herein, a "reference sequence" is a defined sequence used as a basis for sequence comparison. A reference sequence can be a subset or the entirety of a specified sequence, for example, as a segment of a full-length protein or a protein fragment. A reference sequence can be, for example, a sequence identifiable in databases such as GenBank and UniProt, as well as other sequences identifiable to one of skill in the art.

[0151] As used herein, the term "amplified" refers to the process of producing multiple copies of a nucleic acid from a single or fewer copy number of a nucleic acid sequence molecule. An amplified nucleic acid can be referred to as an amplicon.

[0152] As used herein, the term "detect" or "detection" refers to the determination of the existence and / or presence of a target nucleic acid in a limited portion of space, including, but not limited to, a sample, a reaction mixture, a molecular complex, and a substrate. As used herein, "detect" or "detection" can include, but is not limited to, the determination of the chemical and / or biological properties of a target, including, but not limited to, its ability to interact with, specifically bind to, or activate other compounds, as well as additional properties discernible by one of skill in the art upon reading this disclosure. Detection can be quantitative or qualitative. Detection is "quantitative" when it refers to, relates to, or involves the measurement of the quantity or amount (also called quantification) of a target or signal, including, but not limited to, analyses designed to determine the amount or proportion of a target or signal. Detection is "qualitative" when it refers to, relates to, or involves the identification of the presence or absence of a target or signal, without relying on the quantity or amount of the target or signal in addition to its presence or absence.

[0153] As used herein, the term "pathogen reduction technology" refers to techniques, strategies, and / or technologies for reducing, eliminating, and / or inactivating pathogens that may be present in a sample. Several pathogen reduction technologies have been developed, including solvent / detergent treatment, phototreatment (with or without photosensitizers), and chemical treatment.

[0154] As used herein, the term "initial aspiration" is a broad term and should be given its ordinary and customary meaning to those of skill in the art (and is not intended to be limited to any particular or dedicated meaning), and can refer to, but is not limited to, the initial aspiration of a sample or a portion thereof from a sample tube or sample container in order to perform nucleic acid analysis on the sample or portion thereof. By way of example and not limitation, as further described herein, the initial aspiration can include aspirating a sample or a portion thereof from a sample tube using a pipettor. For illustrative purposes, as embodied herein, the initial aspiration can include aspirating a sample or a portion thereof from a sample tube or sample container in a sample tube rack at an aspiration position within an automated system for screening samples of donor blood for shipment of donor material for clinical use. Additionally or alternatively, by way of example and not limitation, as further embodied herein, the initial aspiration can include aspirating a sample from a sample loading area of ​​an automated system for screening samples of donor blood for shipment of donor material for clinical use. By way of example and not limitation, after the initial aspiration, nucleic acid analysis can be performed on the sample or a portion thereof. By way of example and not limitation, as further described herein, after the initial aspiration, the aspirated sample or a portion thereof can be transported to a sample preparation area for a sample preparation process, e.g., the sample can be dispensed into a lysis tube on a sample preparation carousel for a lysis process.

[0155] 1. Mode of sampling; In accordance with the disclosed subject matter, as embodied herein, methods and systems can include sampling to obtain a sample from a subject. By way of example and not limitation, the sample can be obtained from a donor or a patient. The sample obtained by sampling can then be prepared and amplified for downstream analysis and detection in accordance with the disclosed subject matter, as further described herein. By way of example and not limitation, sampling can include any suitable method and / or technique for obtaining a sample from a subject.

[0156] 1.1 Sample type In certain embodiments, the present disclosure provides a method for obtaining a sample from a subject. In certain embodiments, the sample is a biological sample, such as a bodily fluid sample. In certain embodiments, the bodily fluid sample is a bodily secretion. Non-limiting examples of bodily fluid and bodily secretion samples include bodily secretions such as blood (e.g., whole blood, lysed whole blood, serum, or plasma), saliva or oral fluid, sweat, tears, mucus, urine, lymph, cerebrospinal fluid, interstitial fluid, bronchoalveolar lavage fluid, or any other sample suitable for analysis using the methods and techniques described herein. In certain embodiments, the bodily fluid sample is intended for clinical use, such as donor blood for transfusion.

[0157] In certain embodiments, the sample is derived from blood obtained simultaneously, for example, before, during, or after the subject's blood donation. For example, but not limited to, blood can be collected in a blood collection tube at the same time as the blood donation is collected in a separate container, for example, a blood donation collection bag, and the blood collected simultaneously with the blood donation can provide a source of the sample described herein. Thus, as described in detail herein, analysis of such a sample indicates whether the simultaneously collected blood donation contains one or more pathogens or infectious agents. Furthermore, shipping of the blood donation can be based, in part or in whole, on the analysis of one or more samples obtained from such simultaneously collected blood.

[0158] The methods and systems described herein can be used for nucleic acid testing of samples, such as bodily fluid samples. For example, without limitation, the methods and systems described herein can be used for screening blood samples generally, regardless of whether the sample is collected at the same time as a blood donation. However, when the methods and systems described herein are used in connection with screening donor blood, such screening can be used in connection with the donation of substances such as plasma, platelets, red blood cells, and whole blood. In certain embodiments, the blood sample screened in connection with the methods and systems described herein is a whole blood sample. In certain embodiments, the blood sample screened in connection with the methods and systems described herein is a lysed whole blood sample. In certain embodiments, the blood sample screened in connection with the methods and systems described herein is a serum sample. In certain embodiments, the blood sample screened in connection with the methods and systems described herein is a plasma sample.

[0159] In certain embodiments, the donor biological sample is whole blood. As used herein, "whole blood" refers to blood (blood containing both fluid and solid components) that has not had any components removed. Transfusion of whole blood, or the red blood cell (RBC) component of whole blood, can effectively increase the patient's RBC count, thereby increasing the amount of available oxygen-carrying hemoglobin and thereby increasing the patient's oxygen-carrying capacity. In addition to oxygen-carrying capacity, whole blood transfusions can be a source of platelets, which aid in blood clotting. In certain embodiments, the clinical use of platelets, through the use of transfusions, can treat thrombocytopenia, certain cancers, aplastic anemia, and bone marrow transplants.

[0160] In certain embodiments, the donor biological sample is lysed whole blood. As used herein, "lysed whole blood" refers to blood that has not had any components removed (blood containing both liquid and solid components), but in which red blood cells have been lysed, for example, by exposure to a buffer containing ammonium chloride, potassium carbonate, and EDTA. The ammonium chloride lyses RBCs and has minimal effect on lymphocytes. The use of lysed whole blood may be relevant to some NAT screening assays, such as for Babesia and Malaria. Babesia and Malaria are parasites that infect and reside within RBCs to evade detection by the host's immune system. Thus, while Babesia and Malaria are typically absent in plasma or serum samples that lack RBCs, they may be detected in lysed whole blood samples due to the lysis of infected RBCs.

[0161] In certain embodiments, the donor biological sample is plasma. Plasma is the aqueous portion of blood that remains after the cellular components of blood are centrifuged and removed. In certain embodiments, plasma may contain albumin, clotting factors, fibrinolytic proteins, immunoglobulins, and other proteins. Products derived from plasma donations can, in certain embodiments, be used to treat bleeding disorders and / or life-threatening trauma / hemorrhage.

[0162] In certain embodiments, the donor biological sample is serum. As used herein, "serum" is the clear portion of blood plasma that does not contain fibrinogen, cells, or any solid components.

[0163] The methods and systems described herein can be used to screen samples from a single individual as well as multiple individuals.

[0164] In consideration of increased efficiency, sample pooling may not be required in the disclosed systems and methods for the purpose of saving time, according to one aspect of the disclosed subject matter. However, according to one aspect of the disclosed subject matter, sample pooling can be used to the extent desired to increase time and / or save money.

[0165] For example, but not limited to, biological samples from multiple individuals can be pooled together to generate pooled samples for NAT-based screening.In certain embodiments, sample pooling will be carried out by mini-pooling from individuals, such as donors, or at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24 samples. In certain embodiments, the number of individual samples pooled is based on a factor of 8, e.g., a pool of 8 samples, a pool of 16 samples, a pool of 24 samples, a pool of 32 samples, a pool of 40 samples, a pool of 48 samples, a pool of 56 samples, a pool of 64 samples, a pool of 72 samples, a pool of 80 samples, a pool of 88 samples, a pool of 96 samples, a pool of 104 samples, a pool of 112 samples, a pool of 120 samples, a pool of 128 samples, a pool of 136 samples, a pool of 144 samples, a pool of 152 samples, a pool of 160 samples, a pool of 168 samples, a pool of 176 samples, a pool of 184 samples, a pool of 192 samples, a pool of 200 samples. In certain embodiments, the number of individual samples pooled is based on a factor of 2, e.g., a pool of 2 samples, a pool of 4 samples, a pool of 6 samples, a pool of 8 samples, a pool of 10 samples, a pool of 12 samples, a pool of 14 samples, a pool of 16 samples, a pool of 18 samples, a pool of 20 samples, a pool of 22 samples, a pool of 24 samples, etc. In certain embodiments, the pooled sample contains blood from about 2 to about 100 different individuals, e.g., about 5 to about 50, about 5 to about 20, about 5 to about 10, or about 10 to about 20 different individuals.In certain embodiments, for example, when the pooled sample is a pooled plasma sample, the pooled sample can contain plasma from 2 to about 10,000 individuals, e.g., from about 2 to about 9,000, from about 2 to about 8,000, from about 2 to about 7,000, from about 2 to about 5,000, from about 2 to about 4,000, from about 2 to about 3,000, from about 2 to about 2,000, from about 2 to about 1,000, from about 2 to about 900, from about 2 to about 800, from about 2 to about 700, from about 2 to about 600, from about 2 to about 500, from about 2 to about 400, from about 2 to about 300, from about 2 to about 200, from about 2 to about 150, from about 2 to about 100, or from about 2 to about 50 individuals.

[0166] According to one aspect of the disclosed subject matter, samples, such as whole blood lysate or plasma samples, can be pooled using onboard hardware (i.e., onboard pooling). By way of example and not limitation, as embodied herein, the same hardware that manages individual sample preparation can be used to create a sample pool, e.g., of whole blood and / or plasma. For example, but not by way of limitation, as embodied herein, individual samples can be introduced into a sample preparation area, e.g., a sample transporter such as a lysing carousel, and two or more individual samples can be designated for pooling. By way of example and not limitation, as embodied herein, individual samples in a sample preparation area designated for pooling can be returned to the beginning of the sample preparation area and pooled together. Processing of the pooled samples can then be performed using the systems and methods described herein for processing non-pooled samples, as further described herein.

[0167] On-board pooling using the same hardware capable of managing individual samples can reduce the need for additional laboratory capital, such as a separate liquid handler or pooler. Additionally or alternatively, the use of on-board pooling can improve laboratory workflow by reducing or eliminating the need for manual pooling of samples and / or the need for external liquid handlers or pooling systems. In certain embodiments, the use of sample pooling can increase sample throughput. By way of example and illustration, but not limitation, a system with a throughput of approximately 150 tests per hour for individual samples can incorporate a pool size of six samples, thereby reducing the number of tests per hour for individual samples from approximately 150 tests per hour to approximately 50 tests per hour for pools of six samples. As embodied herein, the reduction in the number of tests per hour can be the result of a sample preparation device, such as a lysis carousel, being repositioned to pool samples. By way of example and not limitation, as embodied herein, using a pool size of 6 samples, the number of individual samples tested per hour can be approximately 300, which can represent a 100% increase in total throughput.

[0168] As noted above, samples obtained by the disclosed methods can instead be analyzed on an individual basis, allowing for more rapid processing. Indeed, the methods and systems described herein significantly improve time-to-results and overall throughput, enabling such individual donor NAT ("ID-NAT") screening on a scale that is not only different in degree but also in type from currently implemented strategies. These methods and systems not only facilitate faster donor blood screening, but also represent a step change in how such ID-NAT screening is performed, providing entirely new strategies for enhancing donor and patient safety and access. For example, the methods and systems described herein enable both large-scale and small-scale rapid screening of donor blood without the need for pooling and / or transport to a central sample processing facility. This difference in donor blood screening facility operations improves safety and access to blood and blood products. This improvement is particularly evident in resource-limited regions and during large-scale disasters such as earthquakes and hurricanes, as well as in situations where volunteer donation levels have plummeted, for example, during the COVID-19 pandemic.

[0169] 1.2 Sampling method In certain embodiments, a sample, e.g., a biological sample, can be obtained from a subject by any method known in the art. For example, but not limited to, sampling can include obtaining a biological sample from a subject by arterial sampling or venipuncture sampling. Additionally or alternatively, by way of example and not limitation, a sample can be obtained using a swab, e.g., a nasopharyngeal swab.

[0170] In certain embodiments, biological samples are obtained by venipuncture sampling. For example, but not limited to, sampling is performed by inserting a needle through the skin into the lumen of a vein and filling one or more blood collection containers (e.g., blood collection tubes, satellite bags, or plasma exchange bags) with blood from the vein. Typically, sampling is performed by venipuncture sampling, in which an initial volume of blood enters a diversion bag, capturing the skin introduced by the needle insertion and bacteria present on the skin. After a suitable amount of blood enters the diversion bag, the blood is sent to a blood collection container for testing, such as a blood collection tube, or to a storage container, such as a blood collection bag. In certain embodiments, the blood collection tube is commonly called a "vacutainer," which facilitates the aspiration of a predetermined volume of liquid. It is a sterile glass or plastic test tube with a vacuum seal. Sampling can further include labeling the blood collection container with information about the control. In certain embodiments, the blood collection container contains an anticoagulant, such as a powdered anticoagulant or a liquid coagulant, to prevent blood clotting.

[0171] In certain embodiments, the biological sample is centrifuged. For example, without limitation, a blood sample is centrifuged to separate plasma from the remainder of the sample. In certain embodiments, the blood sample is centrifuged to separate plasma from cells of the sample, e.g., red blood cells, platelets, and / or white blood cells. In certain embodiments, serum is physically separated from the remainder of the sample by centrifugation within about two hours from the time of collection. In certain embodiments, the RBC component of whole blood can be prepared by an automated apheresis method that removes blood from a donor, collects the RBCs, and returns the remaining blood and plasma to the donor. Similarly, plasma can be obtained by an automated apheresis method that removes blood from a donor, collects the plasma, and returns the remaining blood to the donor. In certain embodiments, the sample is obtained by diverting the apheresis line prior to the apheresis process to fill one or more blood collection tubes, e.g., vacutainers. Additionally, or alternatively, the sample can be obtained from collected plasma.

[0172] 2. Sample preparation method In accordance with the disclosed subject matter, as embodied herein, methods and systems for rapid screening of samples, e.g., samples of donor blood, include unique sample preparation aspects for isolating nucleic acids from samples, e.g., samples of donor blood. For example, and without limitation, reference is now made to exemplary sample preparation methods and system components contemplated in the disclosed methods and systems.

[0173] As shown in Figure 1, sample preparation is performed prior to amplification and detection of a nucleic acid of interest. In certain embodiments, sample preparation as embodied herein includes isolation of a nucleic acid of interest from a sample. The present disclosure also contemplates that sample preparation may include one or more additional operations performed in conjunction with sample preparation, such as a reagent preparation operation. In certain embodiments, sample preparation does not include a reagent preparation operation.

[0174] The sample preparation aspects described herein can include the use of various suitable sample preparation techniques for isolating nucleic acids. For example, without limitation, sample preparation can incorporate the use of various sample buffers, nucleic acid immobilization techniques (e.g., immobilization on magnetic particles), and / or elution aspects. Thus, while the methods and systems are described herein with reference to exemplary buffers, immobilization techniques, and elution aspects, those skilled in the art will understand that the methods and systems are not limited to only those exemplary buffers, techniques, and aspects.

[0175] As embodied herein, sample preparation methods and system components can be configured to prepare samples for NAT-based screening. By way of example and not limitation, the sample preparation methods and components can be configured to isolate and / or purify nucleic acids in a sample using any suitable sample preparation technique. In certain embodiments, a sample preparation process for use herein comprises a lysis process and a wash process for isolating and / or purifying nucleic acids in a sample. In certain embodiments, the wash process can comprise one or more wash steps and one or more elution steps. In certain embodiments, a sample preparation process for use herein can comprise a pretreatment process. In certain embodiments, a sample preparation process for use herein can comprise an on-board pooling process. In certain embodiments, a sample preparation process for use herein can comprise an on-board pooling process, a lysis process, and a wash process. In certain embodiments, a sample preparation process for use herein can comprise a pretreatment process, a lysis process, and a wash process. In certain embodiments, a sample preparation process for use herein can comprise a pretreatment process, an on-board pooling process, a lysis process, and a wash process. In certain embodiments, a sample preparation process for use herein can comprise a pretreatment process, an on-board pooling process, a lysis process, and a wash process. In certain embodiments, a sample preparation process for use herein can comprise a pretreatment process, an on-board pooling process, a lysis process, and a wash process. In certain embodiments, a sample preparation process can be selected based on the sample or samples to be analyzed. By way of example and not limitation, when analyzing serum, plasma, and / or lysed whole blood samples, the sample preparation process can include a lysis process and a wash process. Additionally or alternatively, when analyzing serum, plasma, and / or lysed whole blood samples, the sample preparation process can include an on-board pooling process, a lysis process, and a wash process. As further described herein, incorporating an on-board pooling process into the sample preparation process can, for example, increase throughput (e.g., number of samples analyzed per unit time). Additionally or alternatively, when analyzing whole blood samples, the sample preparation process can include pretreatment processes, such as a pretreatment lysis process, a lysis process, and a wash process.Additionally or alternatively, when analyzing whole blood samples, the sample preparation process can include a pretreatment process, an on-board pooling process, a lysis process, and a washing process. As further described herein, incorporating an on-board pooling process into the sample preparation process can, for example, increase throughput (e.g., number of samples analyzed per unit time).

[0176] In certain embodiments, a sample preparation process for use herein can include mixing, such as mixing one or more samples with one or more reagents during a pretreatment process and / or a lysis process. In certain embodiments, a sample preparation process for use herein can include mixing during an on-board pooling process. In certain embodiments, a sample preparation process for use herein can include mixing during a pretreatment process, an on-board pooling process, and a lysis process. In certain embodiments, the pretreatment process is a pretreatment lysis process.

[0177] For example, but not by way of limitation, as shown in FIG. 2A , sample preparation methods and system components embodied herein can be configured to perform a sample preparation process 2 that includes, for example, combining an internal control (IC), microparticles (μP), and a protease, such as proteinase K (PK), a sample, and a sample lysis buffer 4, followed by incubation of the PK / lysis / sample combination 5 to promote binding of nucleic acids to the μP. In certain embodiments, as embodied in FIG. 2A , rapid sample preparation process 2 can further include a wash process 6. In certain embodiments, wash process 6 can include contacting the microparticle-bound nucleic acids with a wash solution, such as lysis buffer or water, for a suitable time and suitable number of washes to substantially remove cellular debris and lysis buffer components, such as GITC, that may interfere with subsequent amplification and / or detection operations. For example, without limitation, as embodied in FIG. 2A , the microparticle-bound nucleic acid can be washed three times, first with lysis buffer at room temperature for about 1 minute, and then twice with water, each wash for about 30 seconds (although other suitable durations and temperatures are contemplated, as described in detail herein). As embodied in FIG. 2A , rapid sample preparation process 2 can include a rapid elution operation 7. In certain embodiments, the rapid elution operation can include contacting the washed microparticle-bound nucleic acid with an elution buffer at about 80° C. for about 3 minutes (although other suitable durations and temperatures are contemplated, as described in detail herein), after which the microparticles can be separated, for example, by magnetic transfer of the microparticles or by magnetic retention of the microparticles while the elution buffer containing the eluted nucleic acid is being transferred, and cooled to prepare the nucleic acid for an amplification and detection process, e.g., amplification and detection process 3. As embodied in FIG. 2A , amplification and detection process 3 can be initiated by contacting the eluted nucleic acid with a combination of amplification reagents, referred to herein as a “master mix.” In certain embodiments, the eluted nucleic acids and the "master mix" can be contacted with an activating agent, such as a divalent metal ion, for example, magnesium.In certain embodiments, as discussed in more detail herein, for amplification and detection process 3, the amplification reaction can last for about 20 minutes at 40°C, although other durations, e.g., about 1 minute to about 20 minutes, about 5 minutes to about 20 minutes, or about 10 minutes to about 60 minutes, and other temperatures, e.g., about 25°C to about 60°C, can be utilized. As embodied herein, sample preparation methods and components can implement the sample preparation process. With reference to Examples 5-23, Figures 2A-B, 5-7, and 39-41, as embodied herein, the sample preparation process can be completed in about 10 minutes. For example, without limitation, sample preparation methods and system components can incorporate magnetic microparticle-based capture of total nucleic acids, e.g., as shown in Figure 9, or magnetic microparticle-based direct capture of target nucleic acids, e.g., as shown in Figure 10. For example, without limitation, as embodied in the magnetic bead-based overall nucleic acid capture process shown in Figure 9, certain methods and systems described herein can utilize one or more sample preparation developments outlined in the lower process of Figure 9, combining, for example, protease (PK) and sample lysis buffer incubation, rapid wash, and / or rapid elution, to achieve sample preparation in approximately 10 minutes or less. Individually or in combination, such sample preparation developments significantly reduce overall process time compared to processes utilizing traditional sample preparation, for example, as outlined in the upper process of Figure 9. In an alternative example, as embodied in the direct capture-based target nucleic acid capture process shown in Figure 10, certain methods and systems described herein can utilize one or more rapid sample preparation developments outlined in the lower process of Figure 10, combining, for example, protease (PK), sample lysis buffer, and nucleic acid binding incubation, rapid microparticle binding, rapid wash, and / or rapid elution, to achieve sample preparation in approximately 12 minutes. Individually or in combination, such rapid sample preparation developments significantly reduce overall process time compared to processes utilizing traditional sample preparation, for example, as outlined in the process at the top of Figure 10.Additionally or alternatively, using the sample preparation developments embodied herein, e.g., those described in Figures 9 and 10, the sample preparation process can be completed in about 20 minutes to about 22 minutes for whole blood samples, and about 10 minutes to about 15 minutes, or about 12 minutes to about 16 minutes, or about 12 minutes to about 14 minutes, or about 10 minutes to about 14 minutes, or about 10 minutes to about 13 minutes, or about 10 minutes to about 12 minutes, or even about 10 minutes to about 11 minutes for serum / plasma samples. By way of example and not limitation, the sample preparation process can include a lysis process of about 3 minutes to about 7 minutes, e.g., about 5 minutes or about 6 minutes. In certain embodiments, the sample preparation process can include a wash process, e.g., one or more wash steps and / or one or more elution steps, of about 6 minutes to about 8 minutes. In certain embodiments, the sample preparation process can include one or more sample wash steps of about 1 minute to about 3 minutes, e.g., one or more sample wash steps of about 2 minutes to about 3 minutes. In certain embodiments, sample preparation can include one or more elution steps of about 2 to about 4 minutes, or about 2 to about 5 minutes, for example, one or more elution steps of about 3 minutes. In certain embodiments, the sample preparation process includes a lysis process of about 4 to about 8 minutes and a wash process of about 5 to about 9 minutes. In certain embodiments, the wash process includes a first wash step, a second wash step, a third wash step, and an elution step.

[0178] In certain embodiments, the sample preparation process can begin by aspirating a sample from a sample container, e.g., from a sample container in a sample tube rack in the aspiration position or from a sample container in a sample loading area, and can end by dispensing eluate into a container of the amplification and detection system. In certain embodiments, the sample preparation process can be completed in about 12 minutes to about 16 minutes, about 12 minutes to about 15 minutes, about 13 minutes to about 15 minutes, or about 13 minutes to about 14 minutes. In certain embodiments, the sample preparation process can be completed in about 12 minutes, about 13 minutes, about 14 minutes, or about 15 minutes. In certain embodiments, the sample preparation process can be completed in about 840 seconds, or about 14 minutes.

[0179] For example, as embodied herein with reference to Figures 2A and 9, methods and components described herein for sample preparation can utilize a sample lysis buffer containing a protease to reduce overall sample preparation time. Additionally or alternatively, methods and components described herein for rapid sample preparation can utilize a sample lysis buffer containing a protease and microparticles for total nucleic acid capture to reduce overall sample preparation time. Additionally or alternatively, methods and components described herein for rapid sample preparation can utilize a sample lysis buffer containing a protease and microparticles for target nucleic acid capture to reduce overall sample preparation time, for example, as embodied herein with reference to Figures 2A and 10.

[0180] Additionally or alternatively, systems and methods can include integrated process pathways for lysis, washing, and elution for different sample types, for example, as embodied herein with reference to FIG. 2B and Examples 5-23. By way of example and not limitation, different sample types (e.g., lysed whole blood, plasma, serum, etc.) can be processed in the same manner along a sample preparation process pathway, such as that shown in FIG. 2B, regardless of the specific nucleic acid analysis to be performed thereafter. This technique, among other benefits, improves the efficiency of sample preparation and the flexibility of the overall method and system by providing the ability to modify the specific nucleic acid analysis to be performed after sample preparation has begun. As shown in FIG. 2B, the integrated process pathway can begin by aspirating a sample from a sample container and contacting the sample with a sample lysis buffer in the presence of microparticles to allow binding of nucleic acids to the microparticles. In certain embodiments, the sample aspirated and contacted with the lysis buffer is a plasma sample, a serum sample, or a lysed whole blood sample. In certain embodiments, the plasma, serum, or lysed whole blood sample will have undergone offline processing prior to aspiration. For example, the whole blood can be centrifuged offline to produce a plasma or serum sample, as described herein, or the whole blood can be treated with an RBC lysing solution to produce a lysed whole blood sample, as described below. In certain embodiments, the sample preparation time for a plasma or serum sample can be about 15 minutes, e.g., about 14 minutes. In certain embodiments, the sample preparation time for a whole blood sample can be about 22 minutes, e.g., about 20 minutes. The volume of the sample aspirated and contacted with the sample lysis buffer in the presence of microparticles can vary, for example, depending on the type of sample. In certain embodiments, the volume of the sample aspirated and contacted with the sample lysis buffer in the presence of microparticles can range from about 50 μL to about 2000 μL. In certain embodiments, for example, for a plasma sample, the volume of the aspirated sample is about 1000 μL, and for lysed whole blood, the volume is about 150 μL. In certain embodiments, the sample contacted with the sample lysis buffer in the presence of microparticles can be incubated at a temperature of about 60°C for about 3 minutes to about 7 minutes, e.g., about 5 minutes to about 7 minutes, or about 5 minutes to about 6 minutes.In certain embodiments, the integrated process path involves the transfer of the microparticle-bound nucleic acid to a first wash buffer. In certain embodiments, the wash buffer can be a sample lysis buffer. In certain embodiments, approximately 500 μL of sample lysis buffer is used as the first wash buffer, although other suitable buffers and depositions are contemplated by the methods and systems described herein. In certain embodiments, the microparticle-bound nucleic acid is washed for approximately 96 seconds in the first wash, although other suitable wash durations are contemplated by the methods and systems described herein. In certain embodiments, the integrated process path involves the transfer of the microparticle-bound nucleic acid to a second wash buffer. In certain embodiments, the second wash buffer can be water. In certain embodiments, approximately 250 μL of water is used as the second wash buffer, although other suitable buffers and depositions are contemplated by the methods and systems described herein. In certain embodiments, the microparticle-bound nucleic acid is washed for approximately 24 seconds in the second wash, although other suitable wash durations are contemplated by the methods and systems described herein. In certain embodiments, the integrated process pathway involves the transfer of the microparticle-bound nucleic acid to a third wash buffer, which in certain embodiments may be water. In certain embodiments, approximately 110 μL of water is used for the third wash with the third wash buffer, although other suitable buffers and depositions are contemplated by the methods and systems described herein. In certain embodiments, the microparticle-bound nucleic acid is washed in the third wash with the third wash buffer for approximately 24 seconds, although other suitable wash durations are contemplated by the methods and systems described herein. In certain embodiments, the integrated process pathway involves the transfer of the microparticle-bound nucleic acid to an elution buffer, which in certain embodiments may include 5 mM PO4. In certain embodiments, approximately 50 μL of elution buffer is used for elution, although other suitable buffers and depositions are contemplated by the methods and systems described herein. In certain embodiments, the microparticle-bound nucleic acid is contacted with the elution buffer at approximately 80° C. for about 3 to about 4 minutes, e.g., about 192 seconds, although other suitable elution durations are contemplated by the methods and systems described herein.Once particulates have been removed, the eluate prepared by the integrated process pathway can be used in one or more amplification and / or detection processes for nucleic acid analysis according to embodiments described herein.

[0181] Additionally or alternatively, as embodied herein, and with reference to FIG. 2B and Examples 5-23, systems and methods can include independent sample preparation techniques for each sample, thus eliminating the need for batch processing. As used herein, batch processing refers to processing multiple samples without (1) the ability to prioritize samples within a group, (2) the ability to prioritize new samples over samples already being processed, and / or (3) the ability to change nucleic acid analyses associated with a particular sample, e.g., specific amplification reactions performed on nucleic acids isolated by sample preparation, after sample preparation has begun. As described in detail herein, the elimination of batch processing provides greater flexibility for prioritizing specific samples and / or specific nucleic acid analyses, improving the overall efficiency of blood monitoring.

[0182] Additionally or alternatively, as embodied herein with reference to Figures 4 and 9 and Examples 3 and 5-23, the sample preparation systems and methods disclosed herein can capture total sample nucleic acids during the lysis step, facilitating capture of low-concentration nucleic acids. For illustrative purposes, and not by way of limitation, with reference to Example 3 and Figures 2A-2B, the systems and methods can utilize microparticles to capture total sample nucleic acids. As embodied herein, the microparticles can include CuTi-coated microparticles. Additionally, or alternatively, the microparticles can be combined with a protease and a sample lysis buffer to reduce the time required to capture total nucleic acids. In certain embodiments, the sample preparation process of the present disclosure can capture total sample nucleic acids using microparticles, such as CuTi-coated microparticles.

[0183] Additionally or alternatively, with reference to Example 4, Figures 2A-2B, and 10 below, the systems and methods can directly capture target nucleic acids using capture oligonucleotides immobilized on microparticles to facilitate isolation of only the target nucleic acid. Additionally or alternatively, the microparticles can be combined with a protease and a sample lysis buffer to reduce the capture time of the target nucleic acid. In certain embodiments, the sample preparation process of the present disclosure can capture only the target nucleic acid using capture oligonucleotides immobilized on microparticles.

[0184] Additionally or alternatively, systems and methods as embodied herein can mix and / or transfer microparticles under magnetic force to enhance mixing and reaction efficiency, e.g., as described in Example 2 and Figures 15-17 and 42. By way of example and not limitation, the type of magnet providing the magnetic force can be an electromagnet, e.g., a fixed electromagnet, or a movable permanent magnet, as disclosed herein. This technique, in embodiments using microparticles, can shorten capture time of total or target nucleic acids, wash time of such nucleic acids bound to microparticles, and / or facilitate transfer to an elution solution.

[0185] For purposes of example and not limitation, an exemplary sample preparation process for a whole blood sample is shown in FIG. 77. In certain embodiments, the sample preparation process for a whole blood sample can include a pretreatment process. For example, as embodied herein, in step 7801, a whole blood sample can be added to a container. From steps 7801 through 7803, the whole blood sample can be lysed, for example, using a buffer containing ammonium chloride, potassium carbonate, and EDTA, as further described herein. From steps 7803 through 7805, the lysed whole blood can be mixed with a lysis buffer and a reagent, for example, a protease, a microparticle, for example, CuTi microparticles, and / or an internal control. As further described herein, in step 7805, nucleic acids of interest can be bound to the microparticles. As embodied herein, steps 7801 through 7805 can be performed on a sample transporter, for example, a lysis carousel, as further described herein. From steps 7805 through 7807, the sample can be washed to purify nucleic acids from the sample. For example, as embodied herein, the sample can be washed three times. As embodied herein, the first wash can be performed to remove cellular debris from the sample and / or to remove materials that may be weakly bound to the particles. As embodied herein, a second and third wash can be performed to remove lysis buffer and / or proteases from the sample. As further embodied herein, after washing, in step 7809, the sample can be eluted to capture nucleic acids previously bound to the microparticles. For example, the microparticles can be exposed to an elution buffer, as further described herein. By way of example and not limitation, as embodied herein, the wash and elution steps 7807 and 7809 can be performed using a wash vessel and a wash track, as further described herein.

[0186] 2.1 Sample dissolution process By way of example and not limitation, the methods and systems of the disclosed subject matter can include a lysis process, i.e., a sample lysis process. In certain embodiments, the sample lysis process includes combining one or more biological samples, e.g., pooled or non-pooled samples, with a sample lysis buffer. In certain embodiments, the sample lysis buffer is a solution adapted to disrupt the membranes or walls of pathogens, infectious agents, and / or cells present within the sample and release the contents of the pathogens, infectious agents, and / or cells, e.g., nucleic acids present within the pathogens, infectious agents, and / or cells.

[0187] As described in detail herein, the introduction of a sample lysis buffer into a sample facilitates the release of nucleic acids from pathogens, infectious agents, and / or cells in the sample. The introduction of such a sample lysis buffer is distinct from the preparation of a "sample type" referred to herein as "lysed whole blood." As described herein, lysed whole blood is a sample type of the present disclosure in which RBCs are lysed by exposure to a lysis buffer, e.g., an RBC lysis solution (e.g., a buffer containing ammonium chloride, potassium carbonate, and EDTA), during a pretreatment lysis process. While such an ammonium chloride-containing buffer lyses RBCs, such a buffer has minimal effect on lymphocytes and would therefore not fall within the scope of "sample lysis buffer" as used herein.

[0188] 2A and 9, the methods and systems of the disclosed subject matter can achieve, among other advantages, improved time to results and improved throughput per unit size due, at least in part, to the use of a particular sample lysis buffer to perform rapid sample preparation. In certain embodiments, the sample preparation process is accomplished by contacting the sample with a sample lysis buffer that includes both: (1) a solution, e.g., a conventional sample lysis buffer, adapted to disrupt the membranes or walls of pathogens, infectious agents, and / or cells present in the sample and release the contents of the pathogens, infectious agents, and / or cells; and (2) a protease, e.g., proteinase K, traditionally used in combination with a separate pre-lysis solution to inactivate nucleases (enzymes that degrade nucleic acids released during exposure of the sample to the sample lysis buffer) and degrade proteins covalently or non-covalently bound to nucleic acids, e.g., proteins covalently bound to HBV DNA. In certain embodiments, the addition of a protease to the sample is optional, such as proteinase K. For example, but not by way of limitation, the sample preparation process can be accomplished by contacting the sample with a sample lysis buffer (e.g., in the absence of a protease, such as proteinase K).

[0189] Additionally or alternatively, the sample lysis process utilizes a conventional sample lysis buffer, e.g., a protease-free buffer, as shown, for example, in the top process pathway of Figure 9. For example, such sample preparation methods and components can utilize preconditioning of the sample by contacting the sample with a preconditioning solution containing a protease that inactivates nucleases and degrades proteins that are covalently or non-covalently bound to nucleic acids, e.g., proteins that are covalently bound to HBV DNA, prior to contacting the sample with the conventional sample lysis buffer.

[0190] Additionally or alternatively, for example, the methods and systems shown in Figures 2-3 and 5-6, i.e., the present disclosure relates to methods and systems configured to initiate contact of a sample with a sample lysis buffer in what are referred to as "pre-lysis systems." In certain embodiments, a system configured to subsequently incubate the sample in the presence of a sample lysis buffer is referred to herein as a "lysis system." Thus, as used herein, a pre-lysis system generally involves combining a sample from a donor (or pool of donors) with a sample lysis buffer and a microparticle-containing reagent, followed by lysis and nucleic acid capture within the sample lysis system. Thus, the use of "pre-lysis" in the context of these systems differs from the use of "preconditioning" in the context of solutions containing proteases for the purpose of inactivating nucleases and degrading proteins covalently or non-covalently bound to nucleic acids.

[0191] Additionally or alternatively, a sample lysis buffer for use in the present disclosure can include one or more of the following components: a protease, a surfactant, a protein denaturant, and a buffer. In certain embodiments, a sample lysis buffer for use in the present disclosure includes a protease, a surfactant, a buffer, and a protein denaturant. In certain embodiments, the protease is optional.

[0192] Additionally or alternatively, a protease is an enzyme that degrades or inactivates one or more nucleases and / or degrades proteins covalently or non-covalently bound to nucleic acids. In certain embodiments, the protease is a serine protease. In certain embodiments, the protease is proteinase K.

[0193] In certain embodiments, the surfactant may be nonionic, anionic, and / or zwitterionic. Non-limiting examples of surfactants include Triton-X, such as Triton X-100 (2-[4-(2,4,4-trimethylpentan-2-yl)phenoxy]ethanol) or Triton X-114 (2-[2-[4-(2,4,4-trimethylpentan-2-yl)phenoxy]ethoxy]ethanol), Tween (polyoxyethylene sorbitan monolaurate), such as Tween-20 (polyoxyethylene (20) sorbitan monolaurate) or Tween-80 (polyoxyethylene) (80) sorbitan monolaurate), sodium dodecyl sulfate (SDS), octylthioglucoside, octylglucoside, and 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS). In certain embodiments, the surfactant is a non-ionic surfactant, such as Tween.

[0194] In certain embodiments, the sample dissolution buffer has a pH of about 5.5 to about 8.0, for example, about 6.0, about 6.5, about 7.0, or about 7.5. In certain embodiments, the pH of the sample dissolution buffer depends on the sample to be lysed. For example, but not limited to, the pH of the sample dissolution buffer may be 6.0. In certain embodiments, the pH of the sample dissolution buffer may be about 7.8.

[0195] In certain embodiments, the sample lysis buffer comprises a chaotropic agent, a buffer, and a surfactant. Non-limiting examples of chaotropic agents include guanidinium salts (e.g., guanidinium thiocyanate, guanidinium hydrochloride, guanidinium chloride, and guanidinium isothiocyanate), urea, potassium iodide, perchlorates (e.g., potassium perchlorate), and other types of thiocyanates. In certain embodiments, the chaotropic agent is guanidinium thiocyanate (GITC). In certain embodiments, the lysis buffer can further comprise a protease.

[0196] In certain embodiments, the sample lysis buffer comprises about 2.5 to about 4.7 M GITC and about 2% to about 10% Tween-20. In certain embodiments, for example, a sample lysis buffer for a plasma or serum sample comprises about 4.7 M GITC, about 10% Tween-20, and a pH of about 7.8. In certain embodiments, for example, a sample lysis buffer for a whole blood sample comprises about 3.5 M GITC, about 2.5% Tween-20, and a pH of about 6.0. In certain embodiments, the sample lysis buffer comprises 3.13 M GITC, 6.7% Tween-20, 100 mM Tris, and a pH of about 7.8.

[0197] Additionally or alternatively, the volume of sample lysis buffer added to the sample depends on the volume of the sample. In certain embodiments, about 10 μl to about 1000 μl, e.g., about 100 μl to about 1000 μl, of sample lysis buffer can be added to the sample. In certain embodiments, about 750 μl of lysis buffer can be added to the sample. In certain embodiments, the ratio of the volume of sample lysis buffer to the volume of the sample is about 1:100 to about 100:1. In certain embodiments, the ratio of the volume of sample lysis buffer to the volume of the sample is about 1:1. In certain embodiments, the ratio of the volume of sample lysis buffer to the volume of the sample is about 0.75:1.

[0198] Additionally or alternatively, the sample can be incubated for a time sufficient to promote lysis. For example, but not limited to, the total incubation time for sample lysis can be from about 60 seconds to about 6,000 seconds. In certain embodiments, the total incubation time for sample lysis can be from about 60 seconds to about 5,000 seconds. In certain embodiments, the total incubation time for sample lysis can be from about 60 seconds to about 4,000 seconds. In certain embodiments, the total incubation time for sample lysis can be from about 60 seconds to about 3,000 seconds. In certain embodiments, the total incubation time for sample lysis can be from about 60 seconds to about 2,000 seconds. In certain embodiments, the total incubation time for sample lysis can be from about 60 seconds to about 1,000 seconds. In certain embodiments, the total incubation time for sample lysis can be from about 60 seconds to about 600 seconds. In certain embodiments, the total incubation time for sample lysis can be from about 60 seconds to about 540 seconds. In certain embodiments, the total incubation time for sample lysis can be from about 60 seconds to about 480 seconds. In certain embodiments, the total incubation time for sample lysis can be from about 60 seconds to about 420 seconds. In certain embodiments, the total incubation time for sample lysis can be from about 60 seconds to about 360 seconds. In certain embodiments, the total incubation time for sample lysis can be from about 60 seconds to about 300 seconds. In certain embodiments, the total incubation time for sample lysis can be from about 60 seconds to about 240 seconds. In certain embodiments, the total incubation time for sample lysis can be from about 60 seconds to about 180 seconds. In certain embodiments, the total incubation time for sample lysis can be from about 60 seconds to about 120 seconds. In certain embodiments, the total incubation time for sample lysis can be from about 180 seconds to about 6000 seconds. In certain embodiments, the total incubation time for sample lysis can be from about 180 seconds to about 5000 seconds. In certain embodiments, the total incubation time for sample lysis can be from about 180 seconds to about 4000 seconds. In certain embodiments, the total incubation time for sample lysis can be from about 180 seconds to about 3000 seconds.In certain embodiments, the total incubation time for sample lysis can be from about 180 seconds to about 2000 seconds. In certain embodiments, the total incubation time for sample lysis can be from about 180 seconds to about 1000 seconds. In certain embodiments, the total incubation time for sample lysis can be from about 180 seconds to about 600 seconds. In certain embodiments, the total incubation time for sample lysis can be from about 180 seconds to about 540 seconds. In certain embodiments, the total incubation time for sample lysis can be from about 180 seconds to about 480 seconds. In certain embodiments, the total incubation time for sample lysis can be from about 180 seconds to about 420 seconds. In certain embodiments, the total incubation time for sample lysis can be from about 180 seconds to about 360 seconds. In certain embodiments, the total incubation time for sample lysis can be from about 180 seconds to about 300 seconds. In certain embodiments, the total incubation time for sample lysis can be from about 180 seconds to about 240 seconds. In certain embodiments, the total incubation time for sample lysis can be from about 240 seconds to about 6000 seconds. In certain embodiments, the total incubation time for sample lysis can be from about 240 seconds to about 5000 seconds. In certain embodiments, the total incubation time for sample lysis can be from about 240 seconds to about 4000 seconds. In certain embodiments, the total incubation time for sample lysis can be from about 240 seconds to about 3000 seconds. In certain embodiments, the total incubation time for sample lysis can be from about 240 seconds to about 2000 seconds. In certain embodiments, the total incubation time for sample lysis can be from about 240 seconds to about 1000 seconds. In certain embodiments, the total incubation time for sample lysis can be from about 240 seconds to about 600 seconds. In certain embodiments, the total incubation time for sample lysis can be from about 240 seconds to about 540 seconds. In certain embodiments, the total incubation time for sample lysis can be from about 240 seconds to about 480 seconds. In certain embodiments, the total incubation time for sample lysis can be from about 240 seconds to about 420 seconds. In certain embodiments, the total incubation time for sample lysis can be from about 240 seconds to about 360 seconds.In certain embodiments, the total incubation time for sample lysis can be from about 240 seconds to about 300 seconds. In certain embodiments, the total incubation time for sample lysis can be from about 300 seconds to about 6000 seconds. In certain embodiments, the total incubation time for sample lysis can be from about 300 seconds to about 5000 seconds. In certain embodiments, the total incubation time for sample lysis can be from about 300 seconds to about 4000 seconds. In certain embodiments, the total incubation time for sample lysis can be from about 300 seconds to about 3000 seconds. In certain embodiments, the total incubation time for sample lysis can be from about 300 seconds to about 2000 seconds. In certain embodiments, the total incubation time for sample lysis can be from about 300 seconds to about 1000 seconds. In certain embodiments, the total incubation time for sample lysis can be from about 300 seconds to about 600 seconds. In certain embodiments, the total incubation time for sample lysis can be from about 300 seconds to about 540 seconds. In certain embodiments, the total incubation time for sample lysis can be from about 300 seconds to about 480 seconds. In certain embodiments, the total incubation time for sample lysis can be from about 300 seconds to about 420 seconds. In certain embodiments, the total incubation time for sample lysis can be from about 300 seconds to about 360 seconds. In certain embodiments, the total incubation time for sample lysis can be from about 360 seconds to about 6000 seconds. In certain embodiments, the total incubation time for sample lysis can be from about 360 seconds to about 5000 seconds. In certain embodiments, the total incubation time for sample lysis can be from about 360 seconds to about 4000 seconds. In certain embodiments, the total incubation time for sample lysis can be from about 360 seconds to about 3000 seconds. In certain embodiments, the total incubation time for sample lysis can be from about 360 seconds to about 2000 seconds. In certain embodiments, the total incubation time for sample lysis can be from about 360 seconds to about 1000 seconds. In certain embodiments, the total incubation time for sample lysis can be from about 360 seconds to about 600 seconds. In certain embodiments, the total incubation time for sample lysis can be from about 360 seconds to about 540 seconds.In certain embodiments, the total incubation time for sample lysis can be about 360 seconds to about 480 seconds. In certain embodiments, the total incubation time for sample lysis can be about 360 seconds to about 420 seconds. In certain embodiments, the total incubation time for sample lysis can be about 360 seconds to about 400 seconds. In certain embodiments, the total incubation time for sample lysis can be about 420 seconds to about 6000 seconds. In certain embodiments, the total incubation time for sample lysis can be about 420 seconds to about 5000 seconds. In certain embodiments, the total incubation time for sample lysis can be about 420 seconds to about 4000 seconds. In certain embodiments, the total incubation time for sample lysis can be about 420 seconds to about 3000 seconds. In certain embodiments, the total incubation time for sample lysis can be about 420 seconds to about 2000 seconds. In certain embodiments, the total incubation time for sample lysis can be about 420 seconds to about 1000 seconds. In certain embodiments, the total incubation time for sample lysis can be about 420 seconds to about 600 seconds. In certain embodiments, the total incubation time for sample lysis can be about 420 seconds to about 540 seconds. In certain embodiments, the total incubation time for sample lysis can be about 420 seconds to about 480 seconds. In certain embodiments, the total incubation time for sample lysis can be about 480 seconds to about 6000 seconds. In certain embodiments, the total incubation time for sample lysis can be about 480 seconds to about 5000 seconds. In certain embodiments, the total incubation time for sample lysis can be about 480 seconds to about 4000 seconds. In certain embodiments, the total incubation time for sample lysis can be about 480 seconds to about 3000 seconds. In certain embodiments, the total incubation time for sample lysis can be about 480 seconds to about 2000 seconds. In certain embodiments, the total incubation time for sample lysis can be about 480 seconds to about 1000 seconds. In certain embodiments, the total incubation time for sample lysis can be from about 480 seconds to about 600 seconds. In certain embodiments, the total incubation time for sample lysis can be from about 480 seconds to about 540 seconds.In certain embodiments, the total incubation time for sample lysis can be from about 540 seconds to about 6000 seconds. In certain embodiments, the total incubation time for sample lysis can be from about 540 seconds to about 5000 seconds. In certain embodiments, the total incubation time for sample lysis can be from about 540 seconds to about 4000 seconds. In certain embodiments, the total incubation time for sample lysis can be from about 540 seconds to about 3000 seconds. In certain embodiments, the total incubation time for sample lysis can be from about 540 seconds to about 2000 seconds. In certain embodiments, the total incubation time for sample lysis can be from about 540 seconds to about 1000 seconds. In certain embodiments, the total incubation time for sample lysis can be from about 540 seconds to about 600 seconds. In certain embodiments, the total incubation time for sample lysis can be from about 600 seconds to about 6000 seconds. In certain embodiments, the total incubation time for sample lysis can be from about 600 seconds to about 5000 seconds. In certain embodiments, the total incubation time for sample lysis can be from about 600 seconds to about 4000 seconds. In certain embodiments, the total incubation time for sample lysis can be from about 600 seconds to about 3000 seconds. In certain embodiments, the total incubation time for sample lysis can be from about 600 seconds to about 2000 seconds. In certain embodiments, the total incubation time for sample lysis can be from about 600 seconds to about 1000 seconds. In certain embodiments, the total incubation time for sample lysis can be about 384 seconds. In certain embodiments, the total incubation time for sample lysis can be at least about 60 seconds, at least about 120 seconds, at least about 180 seconds, at least about 240 seconds, at least about 300 seconds, at least about 360 seconds, at least about 420 seconds, at least about 480 seconds, at least about 540 seconds, or at least about 600 seconds.

[0199] In certain embodiments, the sample can be incubated at a temperature of about 37°C to about 60°C, for example, about 50°C to about 60°C. For example, without limitation, the temperature can be about 50°C, about 51°C, about 52°C, about 53°C, about 54°C, about 55°C, about 56°C, 57°C, about 58°C, or about 60°C. In certain embodiments, the sample can be incubated at a temperature of about 50°C to about 60°C.

[0200] Additionally or alternatively, with particular reference to FIG. 2B and Examples 5-23, systems and methods can include an integrated process pathway for lysis, washing, and elution. By way of example and not limitation, the same sample type (e.g., whole blood, plasma, serum, etc.) can be processed in the same manner along a sample preparation process pathway, such as that shown in FIG. 2B, regardless of the specific nucleic acid analysis subsequently performed. Thus, the sample lysis process can begin, for example, by aspirating the sample from a sample container and end with elution of nucleic acids to produce an eluate for subsequent amplification and detection as disclosed herein, or with transfer of the eluate to a container of an amplification and detection system as disclosed herein. This integrated process pathway technique, among other advantages, can provide the ability to modify the specific nucleic acid analysis performed after the initiation of sample preparation, improving the efficiency of sample preparation and the flexibility of the overall method and system.

[0201] 2.2 Washing and elution steps According to another aspect of the disclosed subject matter, the methods and systems herein can include a washing process that includes one or more washing and elution steps. In certain embodiments, the washing process begins with washing of the microparticles in a first washing step and ends with transfer of the eluate to a container of an amplification and detection system. In certain embodiments, the washing process is part of a sample preparation process. For example, but not limited to, the washing process follows a lysis process in a sample preparation process.

[0202] As described in more detail herein below, the wash and elution steps (e.g., each of the wash processes) of the present disclosure provide distinct advantages that, among other advantages, at least partially support the significantly improved time to results and improved throughput per unit size achieved by the methods and systems of the present disclosure. For example, the wash and elution steps can be configured to elute nucleic acids that can be analyzed by any of the various nucleic acid assays described herein for detecting pathogens or infectious agents, thereby allowing for modification of the pathogen or infectious agent that is detected even after the sample has been prepared for amplification and detection.

[0203] In certain embodiments, at least one wash step produces at least one wash, and at least one elution produces at least one eluate. In certain embodiments, the wash and elution steps are performed after the lysis step, as shown in Figures 2A-2B and 79. The wash and elution steps are generally utilized to purify nucleic acids from the sample and to remove cellular debris and / or lysis buffer components, such as GITC, that may interfere with amplification and / or detection operations.

[0204] Various suitable techniques or methods for purifying nucleic acids from a sample can be used in conjunction with the washing and / or elution steps. For example, but not by way of limitation, nucleic acids present in a sample can be isolated by the use of microparticles capable of binding nucleic acids, such as CuTi microparticles, including the nucleic acid of interest. In certain embodiments, direct nucleic acid capture can be used, e.g., using microparticles (e.g., magnetic glass particles) or other solid supports coated with nucleic acids complementary to the target nucleic acid, as shown, for example, in FIG. 4. By way of example and not limitation, the washing and elution steps can be performed on a wash track system with multiple positions loaded with wash vessels.

[0205] In certain embodiments, nucleic acids present with a sample can be isolated by contacting the sample with a lysis buffer, microparticles, e.g., CuTi microparticles, and optionally a protease, e.g., proteinase K. In certain embodiments, nucleic acids present with a sample can be isolated by contacting the sample with a lysis buffer, an internal control (IC) nucleic acid, microparticles, e.g., CuTi microparticles, and optionally a protease, e.g., proteinase K.

[0206] 2.3 Microparticle-based total nucleic acid capture Furthermore, according to the disclosed subject matter, the disclosed methods and systems achieve, among other advantages, significant improvements in time and efficiency without sacrificing sensitivity, thereby resulting in increased throughput per unit size, at least in part due to the use of microparticle-based total nucleic acid capture. Microparticle-based total nucleic acid capture, in certain embodiments, refers to the use of microparticles that can non-selectively bind nucleic acids, thereby enabling the capture of nucleic acids regardless of sequence. As disclosed herein, the microparticles with bound nucleic acids can be washed during one or more washing steps of a washing process to remove non-nucleic acid components of the sample. The washed microparticles can then be exposed to conditions that cause the elution of the bound nucleic acids. Because microparticle-based total nucleic acid capture does not rely on sequence-specific interactions, this may facilitate the capture of low-abundance nucleic acids and / or nucleic acids with similar sequences that may compete for binding in a sequence-specific approach. One type of microparticle that can be used in connection with microparticle-based total nucleic acid capture is copper titanium ("CuTi") microparticles. As disclosed herein, the sample lysis, washing, and elution steps utilize microparticle-based total nucleic acid capture. Exemplary CuTi microparticle-based nucleic acid capture embodiments for use in the present disclosure are provided in U.S. Patent Publication No. 2017 / 0081655, the contents of which are incorporated herein in their entirety.

[0207] As shown in Figure 4, an exemplary microparticle-based total nucleic acid capture embodiment includes the use of CuTi microparticles to bind nucleic acids within a sample, e.g., a lysed sample. In particular, CuTi microparticles can enable rapid purification of nucleic acids compared to conventional embodiments involving multiple organic extraction steps. Further advantages of using CuTi microparticles are described in detail in U.S. Patent No. 10,526,596, which is incorporated herein in its entirety.

[0208] Additionally or alternatively, the microparticles used in connection with the methods of the present disclosure, e.g., CuTi microparticles, have a diameter of about 0.5 to about 50 μm, e.g., about 0.5 μm, about 1.0 μm, about 1.5 μm, about 2.0 μm, about 5.0 μm, about 10.0 μm, about 20.0 μm, about 30.0 μm, about 40.0 μm, or about 50.0 μm. In certain embodiments, the CuTi is present in the CuTi microparticles in a Cu to Ti ratio of about 2:1, e.g., about 3 to about 1, about 2 to about 1, about 1 to about 1, about 1 to about 2, or about 1:3.

[0209] In certain embodiments, microparticles can be used in the methods of the present disclosure at particle volumes of about 1 μl to about 100 μl. For example, and without limitation, microparticles can be used in the methods of the present disclosure at particle volumes of about 5 μl to about 100 μl, about 5 μl to about 50 μl, or about 10 μl to about 50 μl.

[0210] Additionally or alternatively, the microparticles used in connection with the methods of the present disclosure, e.g., CuTi microparticles, can bind to at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% of the nucleic acids, such as DNA and RNA, in a sample.

[0211] Additionally or alternatively, following binding of nucleic acids to microparticles, e.g., CuTi microparticles, used in connection with the methods of the present disclosure, the microparticles are washed using a washing process disclosed herein to remove contaminants and / or undesired materials from the microparticles in the sample. In certain embodiments, the washing process, e.g., the washing and elution step, can include multiple washes. For example, without limitation, the washing process includes at least about two washes, at least three washes, or at least four washes. In certain embodiments, the washing process includes at least about three washes. In certain embodiments, e.g., for use in capturing total nucleic acids, as shown in Example 8, the washing process includes three washes. In certain embodiments, e.g., for use in capturing target nucleic acids, as shown in Example 4, the washing process includes two washes.

[0212] In certain embodiments, microparticles used in connection with the methods of the present disclosure, e.g., CuTi microparticles, are washed in each washing solution for about 10 seconds to about 5 minutes, e.g., about 20 seconds to about 2 minutes, about 20 seconds to about 1 minute, about 20 seconds to about 48 seconds, about 20 seconds to about 30 seconds, about 30 seconds to about 90 seconds, or about 30 seconds to about 50 seconds. In certain embodiments, the microparticles are washed in each washing solution for about 40 seconds. In certain embodiments, at least one of the washing agents comprises a surfactant and / or a protein denaturant. Non-limiting examples of surfactants are described herein.

[0213] Additionally or alternatively, microparticles used in connection with the disclosed methods, such as CuTi microparticles, can be removed from the sample and placed in a wash solution. For example, but not by way of limitation, microparticles used in connection with the disclosed methods, such as CuTi microparticles, can be captured using a magnet and transferred to a well containing a wash solution. By way of example and not limitation, the microparticles can be transferred using a magnetic tip. Additionally or alternatively, in certain embodiments, the microparticles can be transferred using a plunger. Additionally or alternatively, the microparticles can be transferred using a moving magnet or a fixed magnet. In certain embodiments, the microparticles used in connection with the disclosed methods, such as CuTi microparticles, can be captured and placed in a separate wash solution.

[0214] Additionally or alternatively, wash solutions for use in the present disclosure have a pH of about 5.5 to about 8.0, e.g., about 6.0, about 6.5, about 7.0, or about 7.5. In certain embodiments, the pH of the wash solution depends on the sample. For example, but not limited to, the pH of the wash buffer may be 6.0. In certain embodiments, the pH of the wash solution may be about 7.8.

[0215] Additionally or alternatively, a volume of about 10 μl to about 500 μl of wash solution can be used for each wash, for example, but not limited to, a volume of about 100 μl to about 500 μl of wash solution, e.g., about 250 μl, can be used for each wash.

[0216] In certain embodiments, at least one of the washes is performed using a lysis buffer. In certain embodiments, one of the washes is performed using a wash solution, e.g., a first wash solution, comprising about 2.5 M to about 4.7 M GITC, about 2% to about 10% Tween-20, and a pH of about 5.5 to about 8.0. In certain embodiments, one of the washes is performed using a first wash solution for washing plasma or serum samples comprising about 4.7 M GITC, about 10% Tween-20, and a pH of about 7.8. In certain embodiments, one of the washes is performed using a first wash solution for washing whole blood samples comprising about 3.5 M GITC, about 2.5% Tween-20, and a pH of about 6.0. In certain embodiments, one of the washes is performed using a first wash solution for washing a whole blood sample comprising about 3.13 M GITC, about 6.7% Tween-20, 100 mM Tris, and a pH of about 7.8. In certain embodiments, at least one of the washes is performed using water. For example, without limitation, the first, second, and third washes can be performed using water. In certain embodiments, the second and third washes can be performed using water. In certain embodiments, the first wash is performed using a lysis buffer. In certain embodiments, the first wash is performed using a lysis buffer, and the second and third washes are performed using water. Additionally or alternatively, the bound nucleic acid can be subsequently eluted from the microparticles, e.g., CuTi microparticles, used in connection with the disclosed methods after washing. For example, but not limited to, the microparticles, e.g., CuTi microparticles, used in connection with the disclosed methods can be captured from a final wash solution, e.g., the third wash solution, and placed in an elution buffer. In certain embodiments, elution of nucleic acid from the microparticles, e.g., CuTi microparticles, used in connection with the disclosed methods using an elution buffer produces an eluate containing the pre-bound nucleic acid. In certain embodiments, the elution buffer comprises about 5 mM to about 10 mM phosphate and has a pH of about 7.5 to about 9.0. In certain embodiments, the elution buffer comprises a 5 mM PO4 solution. In certain embodiments, the elution step comprises eluting the microparticles with bound nucleic acid in a 5 mM PO4 solution for 80 minutes. o C, for example, for about 3 minutes.

[0217] Additionally or alternatively, microparticles used in connection with the methods of the present disclosure, e.g., CuTi microparticles, are incubated in the elution buffer for about 1 minute to about 10 minutes, e.g., about 2 minutes to about 9 minutes, about 3 minutes to about 8 minutes, about 2 minutes to about 4 minutes, or about 3 minutes to about 4 minutes. In certain embodiments, microparticles used in connection with the methods of the present disclosure, e.g., CuTi microparticles, are incubated in the elution buffer for about 3 minutes. In certain embodiments, microparticles used in connection with the methods of the present disclosure, e.g., CuTi microparticles, are incubated in the elution buffer for about 200 seconds or less, e.g., about 192 seconds or less. In certain embodiments, microparticles used in connection with the methods of the present disclosure, e.g., CuTi microparticles, are incubated in the elution buffer for about 60 seconds or less. o C~about 100 o C, for example, about 70 o C~about 90 o C, or about 75 o C ~ approx. 85 o In certain embodiments, the microparticles used in connection with the methods of the present disclosure, e.g., CuTi microparticles, are incubated in an elution buffer at a temperature of about 80°C. oThe resulting eluate is incubated in an elution buffer at a temperature of 30°C. The resulting eluate can then be used in a subsequent amplification reaction, or in certain embodiments, in any of the various nucleic acid assays described herein to detect pathogens or infectious agents. In certain embodiments, the resulting eluate has a volume of about 5 μL to about 500 μL, e.g., about 10 μL to about 250 μL, or about 10 μL to about 100 μL.

[0218] While the exemplary embodiments described above refer to the use of CuTi microparticles, the methods and systems of the present disclosure incorporate a wide variety of particles and / or solid supports in addition to or as an alternative to CuTi microparticles to facilitate the isolation of nucleic acids, e.g., target nucleic acids. For example, without limitation, the methods and systems of the present disclosure can utilize particles, e.g., microparticles, and / or solid supports comprising or coated with a wide variety of metal oxides (see, e.g., U.S. Pat. No. 6,936,414, incorporated herein by reference in its entirety). However, the present disclosure is not limited to a particular metal oxide. In certain embodiments, the metal or metal oxide is AlTi, CaTi, CoTi, Fe2Ti, Fe3Ti, MgTi, MnTi, NiTi, SnTi, ZnTi, Fe2O3, Fe3O4, Mg, Mn, Sn, Ti, or Zn (e.g., in anhydrous or hydrated form). Furthermore, in certain embodiments, the particles and / or solid surfaces are composed of organic polymers, such as polystyrene and its derivatives, polyacrylates and polymethacrylates and their derivatives, or polyurethanes, nylons, polyethylenes, polypropylenes, polybutylenes, and copolymers of these materials. In certain embodiments, the particles are polysaccharides, particularly hydrogels, such as agarose, cellulose, dextran, Sephadex, Sephacryl, chitosan, inorganic materials, such as glass, or further metal oxides and metalloid oxides (e.g., oxides of formula MeO, where Me is selected from, for example, Al, Ti, Zr, Si, B, particularly Al2O3, TiO2, silica, and boron oxide), or metal surfaces, such as gold.

[0219] Additionally or alternatively, the particles are magnetic (e.g., paramagnetic, ferrimagnetic, ferromagnetic, or superparamagnetic), while the methods and systems of the present disclosure are non-magnetic. In some embodiments, the particles and / or solid surfaces can have planar, acicular, cubic, tubular, fibrous, columnar, or amorphous shapes, although other geometric shapes are contemplated.

[0220] 2.4 Direct capture of target nucleic acids by microparticles As further disclosed herein, the disclosed methods and systems, among other advantages, enable increased throughput per unit size through significant improvements in time and efficiency without sacrificing sensitivity, based at least in part on the use of microparticle-based direct capture of target nucleic acids during sample lysis, washing, and elution steps. As shown in Figure 4, microparticle-based direct capture of target nucleic acids can include the use of capture oligonucleotides, i.e., oligonucleotides complementary to a target nucleic acid of interest, bound to microparticles to capture the target nucleic acid in a sequence-selective manner. Thus, direct capture of target nucleic acids differs from other systems that capture total nucleic acids without sequence selection. By limiting the nucleic acids captured, and therefore ultimately eluted, to the target nucleic acid, direct capture of target nucleic acids can facilitate amplification and detection of the target nucleic acid due to the absence of potentially competing non-target nucleic acids.

[0221] In certain embodiments, the microparticles have a diameter of about 0.5 to about 50 μm, e.g., about 0.5 μm, about 1.0 μm, about 1.5 μm, about 2.0 μm, about 5.0 μm, about 10.0 μm, about 20.0 μm, about 30.0 μm, about 40.0 μm, or about 50.0 μm.

[0222] In certain embodiments, the microparticles can bind to at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% of the nucleic acids, such as DNA and RNA, in a sample.

[0223] Additionally or alternatively, following binding of the nucleic acids to the microparticles, the microparticles are washed to remove contaminants and / or undesired materials from the microparticles in the sample. In certain embodiments, the washing and elution step includes multiple washes. For example, without limitation, the washing process includes at least about two washes, at least three washes, or at least four washes. In certain embodiments, the washing process includes at least about two washes. In certain embodiments, the washing process includes at least about three washes.

[0224] Additionally or alternatively, at least one of the cleaning agents comprises a surfactant and / or a protein denaturant, non-limiting examples of which are described herein.

[0225] Additionally or alternatively, wash solutions for use in the present disclosure have a pH of about 5.5 to about 8.0, e.g., about 6.0, about 6.5, about 7.0, or about 7.5. In certain embodiments, the pH of the wash solution depends on the sample. For example, without limitation, the pH of the wash buffer may be 6.0. In certain embodiments, the pH of the wash buffer may be about 7.8. In certain embodiments, at least one of the washes is performed using water. For example, the first and second washes may be performed using water. In certain embodiments, the second and third washes may be performed using water. In certain embodiments, the first wash is performed using a lysis buffer, and the second and third washes are performed using water. Additionally or alternatively, a volume of about 10 μl to about 500 μl of wash solution can be used for each wash, for example, but not limited to, a volume of about 100 μl to about 500 μl of wash solution, e.g., about 250 μl, can be used for each wash.

[0226] In certain embodiments, the bound nucleic acid is eluted from the microparticles after washing the microparticles. In certain embodiments, an elution buffer is used to elute the nucleic acid from the microparticles to produce an eluate. In certain embodiments, the elution buffer comprises about 5 mM to about 10 mM phosphate and a pH of about 7.5 to about 9.0.

[0227] Additionally or alternatively, the microparticles are incubated in the elution buffer for about 1 minute to about 10 minutes, e.g., about 1 minute to about 9 minutes, about 1 minute to about 7 minutes, about 1 minute to about 6 minutes, about 1 minute to about 5 minutes, about 1 minute to about 4 minutes, or about 1 minute to about 3 minutes. In certain embodiments, the microparticles are incubated in the elution buffer for about 3 minutes to capture total nucleic acids. In certain embodiments, the microparticles are incubated in the elution buffer for about 2 minutes. In certain embodiments, the microparticles are incubated in the elution buffer for about 2 minutes to directly capture oligonucleotides. In certain embodiments, the microparticles are incubated in the elution buffer for about 192 seconds (i.e., about 3.2 minutes). In certain embodiments, the microparticles are incubated in the elution buffer for about 60 seconds (i.e., about 1 minute). o C~about 100 o C, for example, about 70 o C~about 90 o C, or about 75 o C ~ approx. 85 o In certain embodiments, the microparticles are incubated in the elution buffer at a temperature of about 80°C. o The resulting eluate can be used in a subsequent amplification reaction, or in certain embodiments, in any of the various nucleic acid assays described herein for detecting pathogens or infectious agents.

[0228] 2.5 Magnetic Transfer For example, as further disclosed herein and embodied herein with reference to Example 2, Figures 15-17, and 42, among other advantages, significant improvements in time to results and improved throughput per unit size are achieved by the systems and methods of the present disclosure, at least in part, due to the incorporation of operational steps involving magnetic capture of magnetic particles. Such operational steps include, but are not limited to, mixing; washing; and magnetic particle transfer. Existing methods for mixing and washing magnetic particles generally rely on mechanical stirring and magnetic mixing using moving permanent magnets. However, in certain embodiments, such mixing, washing, and / or transfer can be performed using a system incorporating at least one stationary electromagnet-based magnetic particle capture. For example, but not limited to, one or more separate mixing or washing positions identified herein can be achieved using stationary electromagnet-based magnetic particle capture. Additionally, certain embodiments described herein can be performed using a system incorporating at least one stationary electromagnet-based magnetic particle capture in conjunction with one or more transfer operations at one or more transfer positions within the systems described herein. As will be understood by those skilled in the art, the specific number, orientation, and operational assignment (e.g., mixing, washing, transferring, or eluting) of individual locations can be varied, if desired, and still remain within the scope of the present disclosure.

[0229] The present disclosure, in certain embodiments, contemplates the use of movable permanent magnets and / or fixed electromagnets in various positions to achieve appropriate mixing, washing, and transfer operations. For example, without limitation, movable permanent magnets and / or electromagnets can be positioned on the right side, left side, and / or bottom of the well. In addition, movable permanent magnets and / or electromagnets can be positioned such that one side is higher than the other. The movable permanent magnets and / or fixed electromagnets of the present disclosure can also be used in combination with various well formats known in the art.

[0230] In certain embodiments, opposing movable permanent magnets and / or electromagnets can be positioned to alternately attract magnetic particles to opposite sides of the washing well. However, the position, timing, power, and sequence of the movable permanent magnets and / or electromagnets are completely flexible. For example, but not limited to, a fixed electromagnet can be used to partially collect the magnetic particles and allow the collected particles to fall to the bottom of the well. Additionally or alternatively, the magnetic particles can be collected on the side of the well to facilitate mixing, washing, and / or transfer. Furthermore, the magnetic particles can be collected slowly at lower power or more quickly at higher power.

[0231] Different types of electromagnets are contemplated for use in connection with the embodiments disclosed herein, for example, DC electromagnets can be used, but because DC electromagnets may magnetize magnetic particles, AC electromagnets can be used in combination with or instead of DC electromagnets to avoid creating residual magnetism in the magnetic particles by changing the switching frequency.

[0232] The use of electromagnets in addition to or instead of permanent magnets (or in other mixing, washing, or transfer embodiments) offers several advantages. Mixing or transfer operations during sample preparation typically require moving a permanent magnet in and out of range of the magnetic particles. Using electromagnets eliminates the need for a moving mechanism by simply switching the electromagnet on and off. Furthermore, magnetic particles can be moved from one well to another by sequentially switching adjacent magnets in the array on and off. Incorporating a fixed electromagnet-based particle capture approach can reduce the amount of solid waste generated by eliminating one or more disposable products per test. The use of fixed electromagnets also eliminates specific volume requirements, as well as the need for disposable well covers or movable permanent magnets when transferring magnetic particles from one well to another. This embodiment can also transfer magnetic particles between wells using a movable electromagnet adjacent to the side of the wash vessel, but without contacting the magnetic particles or liquid in the wells, minimizing magnetic particle loss and liquid carryover from source wells to destination wells, thus maximizing assay performance. For example, by providing an eluate containing fewer contaminants from the sample and more nucleic acid.

[0233] In some embodiments, transfer can be achieved using a movable magnet below the well to slide the microparticles within an internal channel at the bottom of the well to collect, transfer, and release the microparticles. Certain embodiments can utilize a stationary electromagnet that selectively switches adjacent magnets on and off to achieve, for example, magnetic particle movement. Other methods of microparticle transfer, such as reverse particle processing, can also be used.

[0234] 2.6 Pretreatment process The present disclosure contemplates, in certain embodiments, the use of pretreatment processes to treat samples. For example, but not by way of limitation, a whole blood sample can be treated with a pretreatment lysis process to generate a lysed whole blood sample prior to further sample processing in the presence of particulates.

[0235] 2.6.1 Pretreatment of whole blood According to one aspect of the disclosed subject matter, the pretreatment process can be a pretreatment lysis process. For example, the pretreatment process can be performed on a sample (e.g., whole blood) before the above-mentioned lysis process (e.g., lysis with a sample lysis buffer in the presence of microparticles) and washing process. In certain embodiments, the pretreatment lysis process includes contacting the whole blood sample with a lysis buffer, e.g., an RBC lysis solution. In certain embodiments, the pretreatment lysis process includes contacting the whole blood sample with a lysis buffer, e.g., at a ratio of whole blood sample to lysis buffer of about 1:1.

[0236] In certain embodiments, a pretreatment process, e.g., a pretreatment lysis process, can be performed on a sample transporter in a sample preparation area. For example, in certain embodiments, a pretreatment process can be performed in a container on the sample transporter as the container is continuously transported along the transport path of the sample transporter between a sample dispensing position and a sample capture and transfer position. In certain embodiments, the pretreatment process and the pretreatment lysis process can be performed on the sample transporter. Additionally or alternatively, as described further herein, in certain embodiments, a pretreatment process, an on-board pooling process, and a lysis process can be performed on the sample transporter. For example, in certain embodiments, after a sample has completed a pretreatment process in a container on the sample transporter, the sample is aspirated from the container and dispensed into another container on the sample transporter, e.g., at the initial sample dispense position, and another aspect of the sample preparation process continues. For example, after pretreatment, the sample can be dispensed into a container at the initial dispense position, and the lysis process can be initiated.

[0237] For purposes of example, and as embodied herein, the pretreatment process can be performed on a sample preparation carousel, such as a lysis carousel. Exemplary operations for the pretreatment process are shown in Table A below. [Table 1]

[0238] In this embodiment, exemplary position L1 corresponds to loading of a lysis tube into a sample preparation carousel, e.g., lysis carousel 6411. In certain embodiments, loading is accomplished by the known "Pick & Place" strategy from a loadable stack. L2 corresponds to a lysis buffer dispense position. L4 corresponds to a sample dispense position. Samples dispensed at this position can be dispensed from a sample container by the known "Sip & Spit" strategy.

[0239] Exemplary positions L5-L16 correspond to incubation and mixing positions. For example, incubation and mixing positions L5-L16 can incorporate the use of resistive heaters, carousel movement, pop-up mixers, lock-step transport, and / or time-prioritized scheduling. In certain embodiments, positions L5-L16 can incorporate incubation in one or more sample lysis buffers. In certain embodiments, samples can be incubated and mixed for about 3 minutes to about 6 minutes, about 4 minutes to about 6 minutes, or about 5 minutes to about 6 minutes. In certain embodiments, samples can be incubated and mixed for about 3 minutes, about 4 minutes, about 5 minutes, or about 6 minutes. In certain embodiments, samples can be...

Claims

1. 1. A method for screening a plurality of samples to determine whether donor blood associated with said samples is acceptable for transfusion, comprising: performing a nucleic acid analysis on each sample of the plurality of samples, the nucleic acid analysis including a sample preparation process, an amplification process, and a detection process; determining results of said nucleic acid analysis, said results including a determination of a predetermined level of nucleic acid from at least one of a plurality of pathogens or infectious agents, each result having a time to result of about 20 minutes to about 45 minutes; and determining whether the donor blood is acceptable for transfusion based at least in part on the results of the nucleic acid analysis.

2. The method of claim 1 , wherein the nucleic acid analysis and the determining of the results of the nucleic acid analysis are performed on an automated system.

3. The automated system occupies 1 m of space ２ 3. The method of claim 2, wherein at least about 140 results are obtained per hour.

4. The volume occupied by said automated system is 1 m ３ 3. The method of claim 2, wherein at least about 70 results are obtained per hour.

5. 2. The method of claim 1, wherein the multiple pathogens or infectious agents are selected from the group consisting of HIV-1, HIV-2, HBV, HCV, parvovirus B19, HAV, WNV, Zika virus, Dengue virus, Chikungunya virus, Babesia, malaria, and HEV.

6. The method of claim 1 , wherein the determination of a level below a predetermined level indicates that the donor blood is acceptable for transfusion.

7. The plurality of pathogens or infectious agents and the predetermined levels are selected from the following: HIV-1 at a predetermined level of at least 1-50 copies / mL; HIV-2 at a predetermined level of at least 1-20 IU / mL; HBV at a predetermined level of at least 1-10 IU / mL; HCV at a predetermined level of at least 1-50 IU / mL; Parvovirus B19 at a predetermined level of at least 1-40 IU / mL; HAV at a predetermined level of at least 1-10 IU / mL; WNV at a predetermined level of at least 1-50 copies / mL; Zika virus at a predetermined level of at least 1-50 copies / mL; dengue virus at a predetermined level of at least 1-50 copies / mL; Chikungunya virus at a predetermined level of at least 1-50 copies / mL; Babesia at a predetermined level of at least 1-20 copies / mL; Malaria at a predetermined level of at least 1-50 copies / mL; and 2. The method of claim 1, wherein the HEV is at a predetermined level of at least 1-20 IU / mL.

8. the multiple pathogens or infectious agents are HIV-1, HIV-2, HCV, and HBV; 2. The method of claim 1, wherein the nucleic acid analysis comprises a multiplex analysis of HIV-1, HIV-2, HCV, and HBV.

9. the plurality of pathogens or infectious agents include Zika virus, WNV, Chikungunya virus, and Dengue virus; the nucleic acid analysis includes multiplex analysis of Zika virus and Dengue virus; The method of claim 1 , wherein the nucleic acid analysis comprises multiplex analysis of Chikungunya virus and WNV.

10. the plurality of pathogens or infectious agents include parvovirus B19 and HAV; 2. The method of claim 1, wherein the nucleic acid analysis comprises multiplex analysis of parvovirus B19 and HAV.

11. 10. The method of claim 1, wherein at least one of the multiple samples of donor blood is a pooled sample.

12. 2. The method of claim 1, wherein the sample preparation process comprises on-board pooling a subset of the plurality of samples to form a pooled sample, and performing the nucleic acid analysis on the pooled sample.

13. 10. The method of claim 1, wherein the sample preparation process comprises preparing an eluate and dividing the eluate into two or more amplification vessels.

14. The method of claim 1 , wherein the sample preparation process comprises a lysis process and a washing process.

15. 15. The method of claim 14, wherein the dissolution process is complete in about 4 to about 8 minutes.

16. The method of claim 14, wherein the cleaning process is completed in about 5 to about 9 minutes.

17. The method of claim 14 , wherein the washing process comprises at least one washing step and an elution step.

18. 10. The method of claim 1, wherein the sample preparation process is completed in about 12 to about 16 minutes.

19. The method of claim 1 , wherein the amplification process and the detection process are performed together in an amplification and detection process.

20. 20. The method of claim 19, wherein the amplification and detection process is completed in about 1 to about 22 minutes.

21. 2. The method of claim 1, wherein the time to result begins with an initial aspiration of the sample to perform the nucleic acid analysis and / or ends with the determination of the result.

22. The automated system comprises: sample loading area; a sample preparation area for carrying out said sample preparation process; A nucleic acid amplification region for carrying out the amplification process; and a nucleic acid detection area for carrying out said detection process; The method of claim 2 , further comprising a sample analysis station comprising:

23. 23. The method of claim 22, wherein the nucleic acid amplification region and the nucleic acid detection region are a single region.

24. 24. The method of claim 23, wherein the single region comprises an amplification and detection system configured to simultaneously perform the amplification and detection processes.

25. 23. The method of claim 22, wherein the sample preparation area comprises a sample preparation carousel and / or a wash and elution system.

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