Devices and methods for extraction-free pathogen testing

Abstract

The present invention provides compositions, devices, methods and kits that enable rapid diagnosis of infectious diseases (including viruses, such as influenza virus and SARS-CoV-2) by extraction-free direct PCR techniques using combined biological samples (e.g., saliva samples and respiratory mucosa samples) in a buffer composition comprising nuclease-free water, antifungal agents, antibiotics and ribonuclease inhibitors. The present invention relates generally to diagnostic methods and, more particularly, to compositions and methods for performing extraction-free pathogen testing and detection.

Description

[Technical field]

[0001] (Technical field) The present invention relates generally to diagnostic methods, and more particularly to compositions and methods for performing extraction-free pathogen testing and detection. [Background technology]

[0002] (background) The rapid global spread of infectious diseases presents major healthcare challenges. For example, the rapid spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has led to a global pandemic, highlighting the importance of rapid and early detection.

[0003] Current detection techniques for many infectious diseases involve the use of polymerase chain reaction (PCR). PCR is a technique used to selectively amplify specific regions of DNA of interest (DNA target). For example, various real-time PCR assays (also called quantitative PCR (qPCR)) for detecting SARS-CoV-2 RNA have been developed worldwide, targeting different viral genes or regions.

[0004] Although current PCR methods enable the detection and diagnosis of infectious diseases, they suffer from drawbacks. One notable drawback is that current approaches rely on an initial step of isolating and purifying nucleic acid from clinical samples as part of the virus testing protocol. This initial nucleic acid separation and purification step (i.e., extraction step), required in conventional methods prior to undergoing PCR, constitutes a major obstacle in this diagnostic process, since it remains manual, laborious and expensive, and further increases the opportunities for accidental contamination and human error.

[0005] Furthermore, the efficacy of PCR-based tests to diagnose SARS-CoV-2 can vary based on the type of sample analyzed (e.g., saliva or nostril nasal swabs), the timing of sample collection relative to the course of infection, and even the subject's behavior prior to sample collection. Recent analyses have shown that for SARS-CoV-2, upper respiratory tract samples have the highest concentrations of viral particles early in the course of infection, which declines after the onset of symptoms. In contrast, lower respiratory tract samples have higher viral loads later in the course of the disease.

[0006] Other viral and non-viral pathogens (e.g., bacteria, fungi, etc.) are also amenable to detection by PCR and related processes. Furthermore, certain pathogens are unstable in representative samples (e.g., saliva), making complete pathogen detection difficult. Summary of the Invention [Means for solving the problem]

[0007] (Summary) The present invention provides compositions and methods for the rapid, extraction-free detection and analysis of nucleic acids in saliva and respiratory mucosa.

[0008] The method of the present invention is applicable to the detection of any pathogen that can undergo PCR amplification, including viruses (e.g., influenza virus and SARS-CoV-2), as well as bacteria and other pathogens. In one aspect, the present invention allows the combination of two different sample types in a single assay, thereby allowing more accurate results throughout the course of an infection. For example, evidence has emerged that the abundance or clearance of SARS-CoV-2 or other respiratory viruses may vary between individuals or at various times during the course of the infection, comparing nasal passages to saliva. Thus, unlike the method of the present invention, tests that rely on a single specimen type may miss positive cases.

[0009] Furthermore, for saliva-based tests, it has recently been discovered that the behavior of the test subject can interfere with the test's ability to positively detect pathogens. For example, saliva samples collected well after the subject has woken up, or after eating, drinking, or brushing their teeth, have presented lower positive detection rates for SARS-CoV-2. By employing two unique sample types, the method of the present invention mitigates the impact that the behavior of the test subject may have on the accuracy of the test. Furthermore, by using combined saliva and respiratory mucosa samples for respiratory infections, the present invention allows for successful detection of the virus using minimally invasive sample collection.

[0010] In another aspect, the present invention provides a stabilization buffer that preserves pathogens in a sample. The buffer described below stabilizes both viruses and bacteria for transport before detection of pathogen nucleic acid. In a preferred aspect, the transport buffer described herein is added to a liquid sample suspected of containing pathogens. The sample is then transported to a laboratory for extraction and testing. Since the buffer composition disclosed herein preserves both viral and bacterial pathogens, multiple pathogen detection assays can be performed on a single sample and / or multiple sample types can be combined for multiple pathogen analysis. The present invention contemplates the use of any sample type, but preferably uses liquid samples (e.g., blood, sputum, saliva, nasal mucosa, cerebrospinal fluid, urine, pus, nipple aspirate, peritoneal fluid, lymph, sweat and tears).

[0011] In a first aspect, the present invention provides a composition for processing combined saliva and respiratory mucosa samples to provide nucleic acid available for subsequent amplification and / or detection (e.g., using next-generation sequencing technology), while eliminating the need for an initial nucleic acid extraction step. The composition of the present invention eliminates the need for pathogen transport media, which typically inhibits PCR. The composition of the present invention includes, for example, a unique buffer for sample transport and preparation, which, when mixed with the sample of interest, allows the preparation of suitable nucleic acid from the sample for direct nucleic acid amplification and analysis without the need for initial nucleic acid extraction (i.e., nucleic acid separation and purification).

[0012] In one aspect, the present invention avoids the traditional approach for pathogen detection, which may include, for example, an RNA extraction step using industrial RNA extraction kits and techniques.Instead, the sample test using the method of the present invention is direct and avoids the extraction step.After the combined sample is provided in a unique buffer composition, the nucleic acid sample can then be used for downstream diagnostic testing based on qPCR, rtPCR, or NGS.The present invention is useful for the detection of DNA or RNA, as required for the detection of target nucleic acid.

[0013] In certain aspects, the present invention includes a kit with all the components necessary to obtain a combined sample, which may preferably be a saliva and mucosa sample. This may include providing a kit to a patient. The subject may provide a sample using the kit's easy-to-use components in the comfort of their own home. Using the proprietary buffer compositions disclosed herein, the sample may be sufficiently preserved and secured so that it may be mailed to a laboratory for analysis.

[0014] For purposes of the present invention, the target nucleic acid may be a human genomic sequence, a human transcript sequence, a pathogen sequence, or a parasite sequence.

[0015] In a preferred aspect, the compositions and methods of the present invention improve upon conventional testing and detection approaches by using a combined saliva and respiratory mucosa sample while simultaneously reducing the number of steps required for sample preparation and testing. As a result, the time required for testing is greatly reduced, resulting in faster turnaround time and delivery of results. Furthermore, because the methods of the present invention test the combined sample using a single assay, the present invention further reduces sample cross-contamination and sample-to-worker infection while also reducing labor costs and consumables.

[0016] In one aspect, the present invention provides a method for detecting a viral infection. In some particular methods, the viral infection is a coronavirus (e.g., severe acute respiratory syndrome coronavirus (e.g., SARS-CoV-2)). However, it should be noted that the method of the present invention is useful for detecting other viral infections.

[0017] The method includes obtaining a combined saliva and respiratory mucosa sample from a patient. In certain aspects, obtaining the combined sample includes collecting saliva from the subject (e.g., by having the patient spit into a suitable collection container) and respiratory mucosa (collected by nasopharyngeal or pharyngeal swab).

[0018] A preferred method further comprises the step of mixing the combined sample with an inventive buffer composition that allows nucleic acid from a biological sample suitable for nucleic acid amplification to be prepared without initial extraction of the nucleic acid. In other words, upon mixing the biological sample with the buffer, certain components present in the buffer allow the nucleic acid from the sample to be sufficiently prepared for subsequent nucleic acid analysis (i.e., amplification by PCR) without the need for typical extraction (separation and purification) steps.

[0019] The buffer compositions used in the methods of the invention generally include nuclease-free water, antifungal solutions, antibiotic solutions, ribonuclease inhibitors, reducing agent solutions, and / or Tris-borate-EDTA buffer solutions. In certain aspects, the buffer composition also functions as a transport medium, in which the combined sample (including any sample collection swabs) is placed directly into a suitable collection vessel containing the buffer composition.

[0020] The method further includes performing one or more PCR assays on the prepared nucleic acid to detect viral nucleic acid, in which case the patient can be diagnosed as infected with the virus.

[0021] The step of carrying out the PCR assay comprises using a viral nucleic acid specific primer-probe set. In certain aspects, the viral nucleic acid specific primer-probe set targets one or more of the N gene, ORF1ab gene, and E gene of the virus. In some embodiments, the step of carrying out the PCR assay comprises using a primer-probe set specific for ribonuclease P (RNP). The extraction method disclosed herein is also useful for detecting human genomic sequences or human RNA sequences, since it is independent of the source of nucleic acid.

[0022] In certain aspects, the method of the present invention further comprises quantifying the viral nucleic acid. For example, the performing one or more PCR assays comprises performing at least one of quantitative PCR (qPCR) and digital PCR (dPCR), which may include droplet digital PCR (ddPCR). In addition to diagnosing the patient as either infected with the virus or not infected with the virus, the method may further comprise determining the severity of the viral infection based on the amount of viral nucleic acid. In some embodiments, the method may further comprise comparing the amount of viral nucleic acid in multiple biological samples obtained from the patient at successive time points and determining disease progression based on the increase or decrease in the amount of viral nucleic acid over time. The method of the present invention may further comprise predicting disease outcome based on the identity or amount of viral nucleic acid. The method of the present invention may also be used to characterize a course of treatment or diagnosis. For example, the results may be used to determine appropriate therapeutic or clinical procedures.

[0023] In another aspect, the present invention provides detection of bacteria using a no-extraction buffer to preserve bacterial DNA and / or RNA for detection. The same buffer is useful for preservation of both viruses and bacteria, thereby allowing detection of viral and bacterial pathogens in the same sample or combination of samples. Thus, in one aspect, the present invention provides a method for stabilizing bacteria and / or viruses in a biological sample for no-extraction testing, for example, by PCR. Thus, the present invention allows for simultaneous detection of viral and bacterial samples. This allows for "all-in-one" testing for viral and bacterial sexually transmitted infections (STIs) (e.g., Chlamydia trachomatis and Neisseria gonorrhea). Furthermore, the buffers disclosed herein stabilize influenza viruses as well as SARS viruses, so that a single test is used to detect influenza and, for example, SARS-CoV-2.

[0024] The present invention also provides a method for extraction-free analysis of nucleic acid. An exemplary method includes providing a vial and obtaining a saliva sample from a subject in the vial. The method further includes obtaining a respiratory mucosal swab sample from the subject. The method includes mixing the saliva sample and the respiratory mucosal swab sample in the vial. Preferably, the combined sample is mixed in the vial with a storage buffer composition, which includes, for example, a buffer nuclease-free water, an antifungal agent, an antibiotic, and a ribonuclease inhibitor. Thus, the method includes directly amplifying nucleic acid in the buffer with a primer specific for a target nucleic acid. The direct amplification is performed without a prior nucleic acid extraction step. After amplification, the method includes analyzing the amplicons generated in the amplifying step to detect the presence of one or more pathogens.

[0025] In certain aspects, a saliva sample is obtained from the subject using a saliva collection aid (SCA) or funnel. The SCA may contain the buffer composition, which is released into the vial. For example, the SCA may contain the buffer composition in an internal pouch or compartment or in a lid, which releases the buffer composition into the vial. In certain aspects, the SCA or funnel includes a lid. The lid may contain the buffer composition, which is released into the vial when the lid is closed. In certain aspects, the SCA or funnel is integrated with the vial. Alternatively, the SCA or funnel may be configured to connect to the vial during saliva collection. In certain aspects, the SCA or funnel is configured such that it can be reversibly connected to the vial.

[0026] Preferably, a respiratory mucosal swab sample is obtained by swabbing the subject's nostrils. In certain aspects, the swab used to obtain the respiratory mucosal sample is attached to a cap that is used to seal the vial. Sealing the vial with the cap may position the swab in the saliva sample such that the saliva sample and the swab sample are combined.

[0027] The present invention also provides kits for carrying out the methods of quantifying nucleic acids, including viral and / or bacterial nucleic acids disclosed herein. In certain aspects, the kits of the present invention include one or more vials, saliva collection aids and / or funnels; buffer compositions (e.g., transport (storage) buffers, primers for amplifying one or more target nucleic acids, and instructions for use. [Brief description of the drawings]

[0028] [Figure 1] FIG. 1 shows a schematic diagram of an extraction-free real-time RT-qPCR test for quantitative detection of nucleic acids derived from SARS-CoV-2 in biological specimens (spit or swab samples) collected and processed with the unique buffer composition of the present invention. [Diagram 2] FIG. 2 shows a sample from a patient suspected of having a viral infection and the loading of the sample into an instrument that can perform one or more assays on the sample to determine whether viral nucleic acid associated with the viral infection is present. [Diagram 3] Figure 3 shows the results of the SARS-CoV-2 qPCR detection protocol performed on paired saliva-only and combined saliva and nasal swab samples obtained from the same patient. [Figure 4] FIG. 4 depicts selected components used in the methods of the present disclosure and provided in certain kits of the present invention. [Diagram 5]FIG. 5 shows selected components of a kit of the invention for detecting a target nucleic acid in a combined sample. [Figure 6] FIG. 6 shows selected components of a kit of the invention for detecting a target nucleic acid in a combined sample. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0029] (Detailed Description) The present invention provides compositions, methods, and kits that allow for rapid diagnosis of infectious diseases by extraction-free direct PCR techniques using combined samples obtained from two or more sources. The present invention also provides stabilizing buffers that allow for extraction-free testing of pathogen nucleic acids, particularly extraction-free testing of multiple pathogens derived simultaneously from one or more sources. Thus, the methods of the present invention include methods for viral testing, bacterial testing, or combinations thereof. Furthermore, the buffers taught herein preserve samples ranging from SARS to influenza to bacteria, so that the samples can be transported without substantial loss of their target pathogens. Finally, the methods of the present invention allow for analysis of the time course of an infectious disease when a pathogen moves from one location to another (e.g., influenza virus or SARS virus moving from nasal passage to pharynx).

[0030] The compositions, methods, and kits of the present invention can be used to process combined biological samples (e.g., saliva and respiratory mucosa) to provide available DNA for subsequent PCR assays while eliminating the need for an initial RNA extraction step. The present invention includes a unique buffer composition for sample transport and preparation that, when mixed with a sample of interest, allows preparation of nucleic acid from the sample that can be used directly for nucleic acid amplification and analysis without the need for initial nucleic acid extraction (i.e., nucleic acid separation and purification). Thus, unlike previous approaches (which include an RNA extraction step using industrial RNA extraction kits and techniques), the direct testing of combined samples of the present invention avoids this process by omitting the extraction step. Instead, after the clinical sample is provided in the unique buffer composition, pathogens can be inactivated by either heating or direct lysis in the buffer. The inactivated sample can then be used for downstream qPCR diagnostic testing.

[0031] As a result, the compositions, methods, and kits of the present invention improve upon conventional pathogen testing and detection approaches by reducing the number of steps required for sample preparation and testing. As a result, the time required for testing is greatly reduced, resulting in faster turnaround time and delivery of results. Furthermore, the present invention further reduces cross-contamination of samples and infection of workers from the samples, while reducing labor costs and consumables. The efficiency and cost savings are expanded by using combined samples according to the methods of the present invention. Tests using combined samples require only a single assay to provide results. Furthermore, because the tests are more sensitive and accurate than existing tests, they reduce the need to provide follow-up tests due to ambiguous or erroneous results.

[0032] It should be noted that the methods described herein can be used to diagnose a variety of infectious diseases, including microbial and viral. However, for simplicity and ease of explanation and examples, the following describes a method for diagnosing SARS-CoV-2 by an extraction-free direct PCR approach.

[0033] SARS-CoV-2 is a virus recently identified as the cause of an outbreak of a respiratory illness (called coronavirus disease 2019 (COVID-19)) with an increasing number of patients with severe symptoms and deaths. Typically, for most respiratory viruses, people are thought to be most contagious when their symptoms are greatest. However, for SARS-CoV-2, there have been reports of asymptomatic spread from infected individuals. The effectiveness of SARS-CoV-2 testing is further complicated as analyses have shown that upper respiratory tract samples have the highest concentrations of viral particles early in the course of infection, which declines after the onset of symptoms. In contrast, lower respiratory tract samples have higher viral loads later in the course of the disease. It is therefore important to detect infection as early and as quickly as possible to monitor the presence of SARS-CoV-2 and to prevent its spread.

[0034] The methods of the present invention provide rapid detection of viral infections (i.e., the presence of the virus in a patient) by reducing the number of steps in sample preparation typically required in conventional virus detection methods that rely on PCR assays. Furthermore, by using combined samples, tests can be performed simultaneously on samples taken from locations harboring high concentrations of viral particles at various times during the course of an infection.

[0035] In general, the workflow includes obtaining a combined biological sample from an individual suspected of being infected. The method of sample collection and the type of sample collected may depend on the specific viral disease to be tested. For example, the combined sample used in the present invention may include one or more bodily fluids and may be collected in any clinically acceptable manner. The bodily fluid sample is generally collected from either a patient who shows signs or symptoms of a viral disease, or a patient who is suspected of having contracted the viral disease due to interacting with others who have tested positive for the disease.

[0036] The body fluid may be, for example, a liquid material from a human or other mammal. Such body fluids include, but are not limited to, mucus, blood, plasma, serum, serum-derived material, bile, blood, maternal blood, phlegm, saliva, expectoration, sweat, amniotic fluid, menstrual fluid, mammary fluid, follicular fluid, fallopian tube fluid, ascites, urine, semen, and cerebrospinal fluid (CSF) (e.g., lumbar CS or ventricular CS). The combined sample may also include a medium that includes cells or biological material. The combined sample may also include a blood clot (e.g., a blood clot obtained from whole blood after serum has been removed). In certain aspects, the combined sample includes two or more of saliva, respiratory mucosa, blood, or semen collected from a subject.

[0037] For SARS-CoV-2, a combined sample typically includes saliva combined with a sample collected by nasopharyngeal or pharyngeal swab. The combined sample is then prepared for subsequent analysis. Preparation of the combined sample includes mixing the sample with a buffer composition that allows for preparation of nucleic acid suitable for nucleic acid amplification from the biological sample without initial extraction of the nucleic acid. In certain aspects, a saliva sample is collected and a swab is placed in the sample for sample preparation. The swab can be squeezed or agitated to extract the sample and mix it with another portion of the combined sample (e.g., saliva).

[0038] As mentioned above, current virus testing approaches rely on an initial step of isolating and purifying nucleic acids from clinical samples as part of their virus testing protocols. For example, the application of qPCR for relative quantification of the RNA of interest is preceded by (1) isolation and purification of total RNA from the sample; (2) elution and possible concentration of the material; and (3) use of purified RNA in a reverse transcription (RT) reaction that produces complementary DNA (cDNA), which is then utilized for the qPCR reaction. This initial nucleic acid isolation and purification step (i.e., extraction step), which is required in current methods before undergoing PCR, constitutes a major obstacle in the diagnostic process, because it remains manual, laborious, and expensive, further increasing the opportunities for accidental contamination and human error.

[0039] The present invention provides a composition for processing a combined sample to provide DNA that can be used for subsequent PCR assays, while eliminating the need for an initial RNA extraction step.For example, a unique buffer composition is used for sample preparation, such that when mixed with a biological sample, the unique buffer composition can prepare nucleic acid from the sample that can be used directly for nucleic acid amplification and analysis without the need for initial nucleic acid extraction (i.e., nucleic acid separation and purification).

[0040] When there is an insufficient amount of nucleic acid for analysis, a common technique used to increase the amount includes amplifying the nucleic acid. Amplification refers to the generation of additional copies of a nucleic acid sequence, and is generally carried out using polymerase chain reaction or other techniques well known in the art (e.g., Dieffenbach, PCR Primer, a Laboratory Manual, 1995, Cold Spring Harbor Press, Plainview, NY). Polymerase chain reaction (PCR) refers to a method by KB Mullis (U.S. Patent Nos. 4,683,195 and 4,683,202, which are incorporated herein by reference) for increasing the concentration of a segment of a target sequence in a genomic DNA mixture without cloning or purification. Primers can be prepared by a variety of methods, including, but not limited to, cloning appropriate sequences and direct chemical synthesis using methods well known in the art (Narang et al., Methods Enzymol., 68:90 (1979); Brown et al., Methods Enzymol., 68:109 (1979)). Primers can also be obtained from commercial sources (e.g., Operon Technologies, Amersham Pharmacia Biotech, Sigma, and Life Technologies). Amplification or sequencing adapters or barcodes, or combinations thereof, are attached to the fragmented nucleic acid. Such molecules can be obtained commercially, for example, from Integrated DNA Technologies (Coralville, IA). In certain embodiments, such sequences are attached to the template nucleic acid molecule using an enzyme (e.g., a ligase). Suitable ligases include T4 DNA ligase and T4 RNA ligase (commercially available from New England Biolabs (Ipswich, MA)). The linkage may be blunt ended or may involve the use of complementary overhanging ends.

[0041] For example, DNA can be synthesized by reverse transcription from viral DNA associated with the virus of interest (if present) in a biological sample, thereby generating complementary DNA (cDNA). As is commonly understood, reverse transcriptase (RT) directs the synthesis of first-strand cDNA using an RNA template and a short primer complementary to the 3' end of the RNA, which can be used directly as a template for amplification (by PCR). This combination of reverse transcription and PCR (RT-PCR) allows for the detection of low abundance RNA in a sample and the generation of the corresponding cDNA, thereby facilitating the cloning of low copy genes. Alternatively, the first-strand cDNA can be made double-stranded using DNA polymerase I and DNA ligase. Many RTs are available from commercial suppliers. The use of engineered RTs improves the efficiency of full-length product formation, ensures that the copy formation of the 5' end of the mRNA transcript is complete, and allows for the amplification and characterization of a faithful DNA copy of the RNA sequence. The use of a more thermostable RT, in which the reaction is carried out at higher temperatures, can be very useful when processing RNA that contains large amounts of secondary structure.

[0042] Digital polymerase chain reaction (dPCR) is an improvement of conventional polymerase chain reaction method that can be used to directly quantify and clonal amplify nucleic acid strand (including DNA, cDNA, or RNA).In dPCR, sample is separated into multiple compartments, and reaction is carried out in each compartment separately, thereby enabling sensitive quantification of target DNA by fluorescence analysis in each compartment, as opposed to a single value for the whole sample as found in standard PCR technology.

[0043] Droplet digital PCR (ddPCR) is a method of dPCR in which the compartments are composed of nanoliter-sized water-oil emulsion droplets, in which PCR reaction and fluorescence detection can be carried out, for example, using droplet flow cytometry. The method for generating and reading droplets for ddPCR has been described in detail elsewhere (see Zhong et al., "Multiplex digital PCR: breaking the one target per color barrier of quantitative PCR", Lab Chip, 11:2167-2174, 2011), but essentially each droplet is like a separate reaction well, and after thermal cycling, the fluorescence intensity of each individual droplet is read in a flow-through instrument such as a flow cytometer, which records the peak fluorescence intensity.

[0044] The compositions and methods of the present invention can be used to detect nucleic acids specific to any virus, but in a preferred embodiment, SARS-CoV-2 is the detection target. Exemplary primers and probes for the detection of SARS-CoV-2 have been disclosed by the Chinese CDC (targeting the N and ORF1ab genes) and the WHO (targeting the E gene) and are provided in Tao S et al., 2020 and Dong I et al., 2020. The compositions and methods of the present invention for the detection of COVID-19 infection using ddPCR of combined saliva and nasopharyngeal samples contemplate using the same primers and probes discussed herein. Furthermore, in some embodiments, the step of performing the one or more PCR assays includes using a primer-probe set specific for ribonuclease P (RNP).

[0045] In addition to diagnosing an individual as infected with the virus, the method of the invention may further comprise determining the severity of the viral infection based on the viral nucleic acid amount in the combined sample.For example, the method of the present invention is useful for assessing viral load, which may directly correlate with disease severity and / or progression.In some embodiments, the method may further comprise comparing the viral nucleic acid amount in multiple combined biological samples obtained from a patient at successive time points, and determining disease progression based on the increase or decrease in the viral nucleic acid amount over time.The method of the present invention may also be used to predict disease outcome and / or severity based on the viral nucleic acid amount.The disease outcome is selected from one or more of intubation, ICU admission, discharge, time to intubation, time to discharge, and death.

[0046] FIG. 1 shows a schematic diagram of an extraction-free real-time RT-qPCR test for quantitative detection of nucleic acid derived from SARS-CoV-2 in a combined biological specimen (spit or swab sample) collected and processed with the unique buffer composition of the present invention. In certain aspects, to collect saliva, the patient simply spits into an acceptable container. A nasopharyngeal swab is used for collection of respiratory mucosa and then placed in the container containing the saliva. The swab can be squeezed or agitated to extract the mucosa sample and mix it with the saliva. The container can contain the unique buffer composition of the present invention or the unique buffer composition of the present invention can be added after the combined sample. In certain aspects, the buffer composition can be used for sample preparation and / or transport medium.

[0047] After collecting the combined sample and providing it with the unique buffer composition, the viral particles can be inactivated either by heating or by direct lysis in the buffer. The inactivated sample can then be used for downstream qPCR diagnostic testing without the need for an additional RNA extraction step (separation and purification) that conventional approaches rely on.

[0048] Rather, the prepared samples can be transferred to a PCR plate (96-well / 384-well) format, where cDNA synthesis by RT and detection by qPCR can be performed. Thus, unlike the widely used approach that includes an RNA extraction step using an industrial RNA extraction kit, direct sample testing avoids this process by omitting the extraction.

[0049] FIG. 4 shows certain components used in the methods of the invention. In certain aspects, one or more of the components may be provided as part of a diagnostic kit, along with instructions for use. As shown, the methods and kits of the invention may include a vial 403. In certain aspects, the vial is provided with a buffer composition 405. The buffer composition is, for example, a viral transport buffer as disclosed herein. In certain kits and methods of the invention, the vial 403 is pre-filled with the buffer composition 405. Alternatively, the buffer composition is added to the vial before or after sample collection.

[0050] Preferably, the vial is at least 1.5 mL so that it can accommodate the saliva sample, the swab sample, and some buffer composition. For example, the sample can be collected in a centrifuge tube (e.g., a screw-cap cryovial). An exemplary vial is provided with a barcode 407, which can be used to track individual vials and / or collected samples. Vials useful in combination with the invention disclosed herein include polypropylene cryovials (e.g., 1.9 mL 2D Barcoded cryovials from NEST Scientific USA, NJ, USA).

[0051] In certain aspects, the vial includes threads 407 or other means for securing a cap, lid, funnel, and / or saliva collection aid. In certain aspects, threads 407 or other securing means may be used to secure a cap 409 to the vial to seal the sample for transport and / or storage. In certain aspects, the cap 409 includes a compartment or pouch 411. Upon securing the cap 409 to the vial, the compartment pierces or otherwise releases buffer composition from inside the compartment or pouch 411 into the vial 403.

[0052] In certain aspects, the methods and kits of the present invention include a means for collecting a saliva sample from a subject. In some methods and kits, the subject simply spits into a provided sterile vial 403. Alternatively, a saliva collection aid (SCA) 413 or funnel 415 is provided to facilitate saliva collection. Exemplary saliva collection aids include, for example, saliva collection aids manufactured by Salimetrics, LLC (Carlsbad, CA). Exemplary funnels include the USP VI polypropylene funnel from NEST Scientific USA. The saliva collection aid 413 or funnel 415 may include a means (e.g., threads 417) for connecting the SCA / funnel to the vial during saliva collection. Alternatively, the funnel or SCA is integrated with the vial to form a single unit.

[0053] Preferably, when provided as a diagnostic kit, the SCA / funnel is pre-attached to the vial. The SCA / funnel may include a means for sealing the combined sample (e.g., a lid or cap). Alternatively, the SCA / funnel may be removed, for example, by a thread and screw attachment means. Once removed, the SCA / funnel may be replaced by a cap or lid to seal the combined sample in the vial.

[0054] The SCA 413 or funnel 415 may include a pouch or compartment that contains a buffer composition (e.g., viral transport buffer) disclosed herein. The pouch or compartment may release the buffer during saliva collection. For example, the pouch or compartment may be integrated into a lid or cap for a funnel / SCA, such as that used in the OME-505 collection kit (DNA Genetek, Inc., Ottawa, Canada). When the lid or cap is closed, the compartment is pierced, thereby releasing the buffer into the vial containing the saliva sample.

[0055] The methods and kits of the invention also include or use a swab for collecting a respiratory mucosal sample. In certain aspects, the swab 419 includes a handle 421 that is held while a sample is being obtained from a subject. The handle 421 may include a break point. After the sample is obtained, the handle is broken at the break point, thereby shortening the length of the handle. Thus, the swab 423 with the shortened handle is short enough to fit within the vial 403. As shown, the level 425 of saliva and / or buffer in the vial is sufficient to cover the swab. However, the level 425 of saliva / buffer does not need to cover the swab. Rather, it is only necessary that the saliva / buffer be in a sufficient amount that the swab and saliva sample can be mixed in the vial.

[0056] Alternatively, or in addition, the swab 421 is coupled to a cap 427. The cap 427 may be coupled to the vial 403 after sample collection to seal the sample for transport, storage, and / or processing. As shown, when the cap 427 is secured to the vial 403, the swab is positioned within the saliva / buffer solution in the vial.

[0057] In a preferred aspect, the buffer composition is provided in a pre-filled vial or as part of another component of the kit (e.g., cap as described herein).By providing a pre-measured volume of the buffer in a manner that can be easily added to the sample by the subject, the exemplary kit of the present invention allows the subject to provide a sample at home.By adding a pre-measured novel viral transport buffer composition of the present invention to the combined sample, the subject can provide the sample at home or any other convenient place, and send the sample by mail to a laboratory for analysis.

[0058] 5 details selected components of a kit of the invention used to detect a target nucleic acid in a sample (e.g., a target nucleic acid indicative of a viral infection). The kit includes instructions including the steps necessary to obtain a combined sample. The instructions outline that a vial 503 is provided to the subject along with a saliva collection tool (e.g., saliva collection aid 505). As shown, in certain kits of the invention, the vial 503 is pre-filled with a viral transport buffer 509 as described herein.

[0059] In the exemplary kit, the subject provides a saliva sample to the vial using a provided saliva collection aid 505. As shown, the saliva collection aid 505 is shaped to fit securely into the opening of the vial 503 to facilitate sample collection.

[0060] The kit also includes a swab 511, which is used to swab the subject's nostrils. The handle of the swab includes a break point 513. After the swab has been used to obtain a sample, the handle is broken off at the break point. The shortened swab is placed into the vial containing the saliva sample and buffer. The vial is then sealed with a cap for storage or transport. In certain aspects, the kit includes materials for the subject to mail the combined sample to a laboratory for analysis.

[0061] In certain aspects, the kit comprises one or more primers, at least one of which is used for amplifying and / or detecting a target nucleic acid in the sample.

[0062] FIG. 6 details selected components of a kit of the invention used to detect a target nucleic acid in a sample (e.g., a target nucleic acid indicative of a viral infection). The kit includes instructions, including the steps necessary to obtain a combined sample. The instructions outline that a vial 603 is provided to a subject. The kit includes a saliva collection aid 605 that can be fixedly connected to the vial 603. Preferably, the kit includes a saliva collection aid 605 pre-connected to the vial 603. The saliva collection aid 605 includes a cap 607. When a saliva sample is provided in the vial, the cap 607 is closed over the saliva collection aid 605. When the cap 607 is closed, a compartment in the cap ruptures to allow viral transport buffer to flow into the vial 603 (which contains the saliva sample).

[0063] The kit also includes a swab 609, which is used to swab the subject's nostrils. As shown, the swab is secured to a sealing cap 611. When the swab sample is to be obtained, the swab 609 is placed into the vial and the cap is secured to the vial 603 using threads. By securing the sealing cap 611 to the vial 603, the swab sample is located within the saliva and buffer solution contained within the vial.

[0064] 2 illustrates a combined sample 102 (e.g., saliva and respiratory mucosa) collected from a patient suspected of having a viral infection, and the loading of the sample into an instrument 200 that can perform one or more assays on the sample to determine whether viral nucleic acid associated with the viral infection is present. As described in more detail herein, the combined sample 102 (saliva and respiratory mucosa) obtained (operation 12) from a patient suspected of having a viral infection (or who has or has been in close contact with one or more patients suspected of having the viral infection) may be contained within a suitable container 104.

[0065] For example, the combined sample can be collected and stored in its own container (e.g., centrifuge tube, e.g., screw-cap cryovial). Preferably, 1.9 ml screw-capped cryovials are used. A funnel or saliva collection aid is used to facilitate saliva collection, and a nasal swab with a proximal breakpoint is used, which allows the swab to be inserted into the tube after use. The advantage of using the same tube for both saliva and nasal swabs is that it facilitates downstream sample acceptance automation, e.g., using a decapper. The screw cap is important to prevent contamination. The standard size of the cryovial allows for direct sample storage without additional sample transfer.

[0066] 2 further illustrates the loading of the combined sample 102 onto a PCR plate 106 where sample preparation (introduction of the sample into a unique buffer and / or PCR mix) may occur, at which point the plate 106 may then be introduced into an instrument 200 capable of performing one or more PCR assays on the sample 102 to determine whether viral nucleic acid associated with the virus is present. In particular, the instrument 200 may be configured to provide any one of the preliminary steps of the method, including, but not limited to, detection of viral RNA, reverse transcription of the RNA to produce cDNA, amplification of the cDNA (operation 16), analysis of data from the amplification step (operation 18), and generation of a report 300 (operation 20) that provides information related to the viral assessment.

[0067] Thus, the instrument 200 is generally configured to detect, sequence, and / or count the target nucleic acid or resulting fragments. In this case, if multiple fragments are present or expected, the fragments may be quantified, for example, by qPCR. The resulting report 300 may include specific data related to the assay, such as patient data (i.e., background information, attributes and characteristics, medical history, follow-up information, etc.), test data (whether the sample tested positive or negative for the virus, and if positive, further endpoints, including disease progression and predicted disease outcome). EXAMPLES

[0068] (Examples of saliva and nasal swabs) The following example provides an exemplary protocol for detecting viral nucleic acid according to the method of the present invention. A combined biological sample (e.g., a combined saliva and respiratory mucosa sample) is obtained, which comprises samples from at least two locations of a subject. Although the following example emphasizes a combined saliva and respiratory mucosa sample, other combined samples are within the scope of the present invention.

[0069] For example, the combined sample used herein may comprise one or more different combinations of bodily fluids.Exemplary bodily fluids include, but are not limited to, mucus, blood, plasma, respiratory mucosa, serum, serum-derived substances, bile, blood, maternal blood, phlegm, saliva, expectoration, sweat, amniotic fluid, menstrual fluid, mammary fluid, follicular fluid, fallopian tube fluid, ascites, urine, semen, and cerebrospinal fluid (CSF) (e.g., lumbar CS or ventricular CS).The combined sample may also comprise a sample that is a medium that contains cells or biological material.The combined sample may also comprise a blood clot (e.g., a blood clot obtained from whole blood after serum is removed).

[0070] Furthermore, as shown in the following examples, some of the combined samples can be obtained using various techniques (e.g., by using swabs and / or direct bodily fluid collection).For many respiratory infections, biological samples are generally collected by nasal or pharyngeal swabs, or in some cases, saliva.In other examples, the sample can include aerosol samples or droplets obtained in the air, or more preferably droplets obtained by coughing or sneezing.

[0071] Example 1 On-site combined saliva and nasal swab sample collection Saliva samples are collected from individuals, for example, by having them spit into the sterile container provided.Saliva collection device includes, for example, a screw-capped Nest 1.9ml cryogenic storage vial (or "Nest tube") with a 10-digit one-dimensional barcode pre-printed on the side and a DATAMATRIX two-dimensional code laser-etched on the bottom, and is used as the container for saliva samples.Saliva collection support funnel (Nest) can be used in series with the Nest vial.

[0072] Nasal swab collection devices include, for example, the oral / nasal swab provided by Nest, which is used to swab the patient's nostrils.The swab with the respiratory mucosa sample is placed inside the Nest tube containing the patient's saliva sample, with the head of the swab facing down.The head of the swab can be agitated or squeezed to release the sample from the swab and / or combine the saliva sample with the swab sample.

[0073] Nasal swabs should be collected under the supervision of a trained healthcare professional designated by the agency overseeing the collection site. The healthcare professional overseeing the collection should clean their hands with an alcohol-based sanitizer or unscented soap and water and wear appropriate PPE (gown, gloves, face mask, and / or face shield). Prior to collection, the patient is presented with instructions (e.g., these instructions recommended by the FDA (https: / / tinyurl.com / nasalswabl-2)). The healthcare professional ensures that all patient information (including name, date of birth, and any additional information required by state reporting regulations) is properly completed prior to collection. The healthcare professional then asks the patient to review the study consent form (provided by Ovation) to decide to participate or not participate in the study. Finally, the healthcare professional scans the pre-printed barcode indicator, links it to the patient information already collected, and then places the indicator into the Nest tube to be used by the patient.

[0074] The HCW removes the cap of the Nest tube and instructs the patient to swab 10 times from their own nostril, breaking the proximal break point of the swab inside the tube. The HCW replaces the cap on the Nest tube, making sure to tighten the cap. If any sample is spilled during this collection process, the HCW wipes the outside of the tube with an alcohol wipe or equivalent to prevent contamination. The samples are then placed in individual bags at room temperature before transport to the laboratory.

[0075] Healthcare workers overseeing the swab sample collection should use an alcohol-based hand sanitizer after handling each patient sample.

[0076] (Receiving and accepting combined samples in the laboratory) The combined samples are transported to the laboratory. Samples are removed from the bag and visually inspected by the receiving supervisor at the receiving desk for any leaks or damage. Samples that pass the supervisor's pre-screening step are moved to a desk used by the receiving team. Samples that do not pass the pre-screening step are set aside for further investigation. The receiving personnel scans the barcode of the Nest tube and looks up the patient information and consent status that is displayed on the computer screen by the Laboratory Information Management System (LIMS). Tubes with complete patient information in the LIMS and no leaks (i.e., eligible samples) are placed in a barcoded 48-format rack. The location of the sample in the rack should match the assigned location in the LIMS. Unqualified samples are placed in a separate barcoded 48-format rack and set aside for further investigation by the receiving supervisor. The rack of samples may then be placed in a platform locker in a holding position at 60 rpm until a Medical Laboratory Scientist (MLS) from the sample preparation team retrieves the samples.

[0077] (Reaction Buffer) As part of the sample preparation, the combined sample is mixed with a unique buffer composition (referred to herein as the combined saliva / mucosa preparation buffer) specifically formulated for the combined saliva and respiratory mucosa sample. Preparation of this saliva / mucosa preparation buffer involves the use of at least the following equipment: a safety cabinet or clean bench (a work area capable of maintaining a sterile environment); sterile, individually wrapped pipettes, pipette tips (e.g., 10 mL and 25 mL); pipette aid; pipettor (1 mL or 200 μL) and corresponding tips; and 50 mL sterile, nuclease-free Falcon tubes. An exemplary saliva / mucosa preparation buffer includes the following reagents / components: · 0.5M Bond-Breaker TCEP solution, (Tris(2-carboxyethyl)phosphine hydrochloride, neutral pH), sterile, DNase-free, RNase-free, and protease-free grade, ThermoFisher Scientific, catalog number 77720, 5 mL; ·RNase inhibitor, human placenta, 40,000 units / ml, sterile, DNase-free ·RNase-free grade, New England Biolabs, Catalog No. M0307L, 10,000 units, 250 μl / tube; · Amphotericin B solution, 250 μg / ml in deionized water, sterile, Sigma-Aldrich, Cat. No. A2942, 100 ml (or a similar antifungal agent at an appropriate concentration to prevent fungal contamination and growth); · Penicillin-Streptomycin Solution, 100×, a 100-fold working concentration mixture of penicillin (10,000 IU) and streptomycin (10,000 μg / ml), sterile, Corning, catalog number 30-002-CI (or a similar antibiotic at an appropriate concentration to prevent bacterial contamination and growth); Nuclease-free water, sterile, Millipore / Sigma, W4502, DNase-free, RNase-free and protease-free grade; and Disinfectant (e.g. 70% ethanol).

[0078] Preparation of the saliva / mucosa preparation buffer is performed in a safety cabinet or clean bench according to standard biological and / or clinical laboratory practices and procedures.

[0079] Preparation of these components includes at least the following steps: cleaning the work surface with an appropriate disinfectant; sterilizing reagent bottles before placing them on the work surface; dispensing 40 mL of nuclease-free water into sterile 50 mL Falcon tubes and storing at room temperature; dispensing 4 ml / tube of amphotericin B (into sterile 5 ml Corning tubes) and storing at -20°C; dispensing 1 ml / tube of penicillin / streptomycin (into sterile Eppendorf tubes) and storing at -20°C; recording lot information and preparation in a laboratory-maintained notebook.

[0080] The preparation of the saliva / mucosa preparation buffer includes at least the following steps: 1. Clean the work surface with an appropriate disinfectant; 2. Sterilize reagent bottles before placing them on the work surface (dispensing everything except RNase inhibitor) 3. For example, to prepare 5 mL of buffer (for 1000 tests): 3.1. Add 4.3 mL of nuclease-free water to a sterile 15 mL Falcon tube; 3.2. Add 400 μL of TCEP; 3.3. Using a sterile pipette, add 50 μl of RNase inhibitor; 3.4. Thaw one tube of amphotericin and one tube of penicillin / streptomycin and use a sterile pipette to aseptically add 200 μL of amphotericin and 50 μL of penicillin / streptomycin to the 15 mL Falcon tube; 4. Record lot information and preparation in a laboratory maintained notebook; 5. Assign appropriate laboratory identification (e.g., lot number); 6. Cap the tube tightly and mix thoroughly by inverting the tube; 7. Withdraw 100 μl of medium for quality control (QC) sample; 8. In that bottle Saliva / Mucosa Reaction Buffer Laboratory ID: (insert appropriate ID for the laboratory (e.g., STB1 for Summit Buffer 1)) DOM: (insert current manufacturing date) Expiration Date: (insert the date one month after the manufacturing date) Store at 2℃~8℃ and label it; 9. Store at 2°C to 8°C and add 5 μl to each test along with 30 μl of combined saliva and respiratory mucosa sample and 5 μl of proteinase K when performing FAST tests; 10.Perform sterility checks.

[0081] Example 2 Preparation of combined saliva / mucosa samples A Medical Laboratory Scientist (MLS) from the sample preparation team retrieves a rack of received samples from the locker and brings the rack to the sample preparation room to prepare them for testing. The MLS brings a prepared 96-well sample preparation plate (SPP) containing 10 μL / well of sample preparation mix (SPM). The SPM contains saliva / mucosa preparation buffer and protease (proteinase K). Specifically, the 96-well SPP contains 10 μL of SPM per well (5 μL saliva / mucosa preparation buffer and 5 μL proteinase K (Promega)) dispensed into each well using a multichannel equalizer or Viaflow (Integra). The caps of the combined samples are decapped using a semi-automated 6-channel decapper (Brooks) or an automated 48 format decapper (Brooks) inside a safety cabinet. If the 6-channel decapper is used, the caps are temporarily placed in its cap carrier rack. Using the E1-ClipTip electronic multichannel (8 channel) equalizer, gently pipette approximately 30 μL of the combination from the tubes in the 48-well rack into a 96-well SPP containing 10 μL of SPM. Two 48-well racks of samples fill one 96-well SPP. Recap the samples (6 at a time if using a 6-channel decapper, or 48 at a time if using an automated 48 format decapper). Thoroughly mix the combined saliva / mucosa samples and SPM by placing the plate on a digital microplate shaker at 500 RPM for 1 minute. Place the plate in a miniAmp 96-well PCR instrument at 95°C for 5 minutes and hold at 4°C. The entire rack of samples is then transported to a temporary sample storage area. Any samples requiring repeat testing are identified from this temporary sample storage area. Only one repeat is allowed. In case of failure, request a new sample. Any remaining samples are stored at -80°C for future use.

[0082] Preparation of PCR reagents and plate configuration (combined saliva / mucosa samples) The plate containing the PCR master mix (herein referred to as the PCR master mix plate (PMMP)) contains 12.5 μL of PCR master mix dispensed into each well of a 96-well or 384-well plate using a multichannel equalizer or Viaflow (Integra). The PCR master mix is ​​composed of 10 μL of Luna Universal Probe One-Step Reaction Mix, 1 μL of Luna Warmstart RT enzyme mix, and 1.5 μL of Nl / RNP primer / probe set. The 1.5 μL of Nl / RNP primer / probe is made by adding 50.25 μL of each 100 μM primer and probe stock to 524 μL of IDTE buffer (pH 7.5) to create a 6.7 μM working stock of Nl primer and a 6.7 μM working stock of RNP primer, and 1.7 μM of FAM-labeled Nl and ATTO-647-labeled RNP probe.

[0083] The molecular team Medical Laboratory Scientist (MLS) places a 96- or 384-well PCR Master Mix Plate (PMMP) into their individual PCR workstation and adds 7.5 μL of the processed combined saliva and respiratory mucosa sample from the combined saliva / mucosa sample preparation step above to each designated well of the PMMP. The processed saliva sample is then mixed with the PCR Master Mix above by pipetting, taking care to avoid creating bubbles. The MLS then adds 7.5 μL of the positive control (IDT synthetic 2019-SARS-CoV-N control, 4000 copies / μL) and negative control (IDT Hs-RPP30 control, 4000 copies / μL) for SARS-CoV-2, as well as a no template control (NTC-water) to the PCR wells designated for those controls (one positive control, one negative control, and NTC per plate) and mixes by pipetting, avoiding creating bubbles. The MLS then places a clear plastic qPCR film over the PMMP, seals the film with a plate sealer, and centrifuges briefly in a plate centrifuge to remove any bubbles.

[0084] (PCR temperature profile (amplification region) (combined saliva / mucosa test)) Place the plate in a Bio-Rad CFX or QuantStudio PCR instrument, open the master file "ST-COV-PCR protocol" and run the following thermocycling conditions: 1. Step 1: 55°C for 10 min, 1 cycle; 2. Step 2: 95°C for 1 min, 1 cycle; and Step 3: 40 cycles of 95° C. for 10 seconds, 60° C. for 30 seconds (plus reading the plate in both the FAM channel for the N1 target and the Cy5 channel for the RNP target).

[0085] (Data Interpretation (BioRad CFX opus 96-well format) (Saliva / Mucous Testing)) Bio-Rad CFX reports Cq values. The Cq value file (csv file) is then exported from the PCR instrument to OvDx LIMS. The interpretation of the Cq values ​​(DETECTED, NOT DETECTED, and INVALID) is exported to OvDx LIMS according to the following criteria: [Table 1]

[0086] If N1 is detected, the result is valid and returns "DETECTED" regardless of the value for RNP. If N1 is not detected and RNP is <35, return a result of "NOT DETECTED". If RNP Cq value >35 and Nl >36, rescue the sample for retesting. After retesting, if the RNP is still >35, the donor should be contacted and another sample should be collected. NaN = Not a Number.

[0087] (Quality Assurance and Batch Release (Saliva / Mucous Membrane Testing)) The Laboratory Supervisor tests the controls including: Positive Control (2019-nCoV_N_Control, IDT), which should be positive for the Nl target but negative for the RNP target; Negative Control (Hs_RPP30 Control), which should be negative for the Nl target but positive for the RNP target; and NTC Control, which should be negative for both the Nl and RNP targets. The Laboratory Supervisor also performs spot testing and evaluates the ratio of positive to negative results. The Medical Director releases the batch after further testing and approves the report.

[0088] (Sample placement after PCR test (saliva / mucosa test)) Samples with INVALID results are identified in the temporary sample storage area (Fume Hood 1) above. Replicate testing is performed on these samples, starting from step III (Preparation of Combined Salivary Respiratory Mucosa Sample). Samples with validated results are stored at -80°C. PCR plates are moved to the waste area (Fume Hood 2) as a biohazard.

[0089] On-site collection of nasal and oropharyngeal swab samples The nasal swab collection device comprises a 1.9 ml Nest tube (used as a container for the nasal swab sample) filled with 1 ml of a unique buffer composition specific for the swab sample (hereafter referred to as Swab Transport Buffer). An oral / nares swab provided by Nest is used to swab the patient's nostrils and then placed inside the Nest tube filled with the Swab Transport Buffer.

[0090] The oropharyngeal swab collection device comprises an oral / nasal swab provided by Nest that is used to swab a patient's oropharynx. The swab with the oropharyngeal sample is placed into the Nest tube described above containing the nasal swab and swab transport buffer.

[0091] Nasal swabs (external nares) and oropharyngeal swabs should be collected under the supervision of a trained healthcare professional designated by the agency overseeing the collection site. The healthcare professional overseeing the collection should clean their hands with an alcohol-based sanitizer or unscented soap and water and wear appropriate PPE (gown, gloves, face mask, and / or face shield). Prior to collection, the patient is presented with written instructions. The healthcare professional ensures that all patient information (including name, date of birth, and any additional information required by state reporting regulations) is properly completed prior to collection. The healthcare professional then asks the patient to review the study consent form (provided by Ovation) to decide to participate or not participate in the study. Finally, the healthcare professional scans the pre-printed barcode indicator, links it to the patient information already collected, and then places the indicator into the Nest tube to be used by the patient.

[0092] The HCW removes the cap of the Nest tube and instructs the patient to swab each nostril 10 times, breaking the proximal break point of the swab inside the tube. The HCW similarly obtains an oropharyngeal swab. The HCW replaces the cap on the Nest tube containing both swabs, ensuring that the cap is tightly screwed on. If any sample is spilled during this collection process, the HCW wipes the outside of the tube with an alcohol wipe or equivalent to prevent contamination. The samples are then placed in individual bags at room temperature before transport to the laboratory.

[0093] Healthcare workers overseeing the swab sample collection should use an alcohol-based hand sanitizer after handling each patient sample.

[0094] (Receiving and Accepting Combined Swab Sampling in the Laboratory) The combined swab samples are transported to the laboratory. Samples are removed from the bag and visually inspected by the receiving supervisor at the receiving desk for any leaks or damage. Samples that pass the supervisor's pre-screening step are moved to a desk used by the receiving team. Samples that do not pass the pre-screening step are set aside for further investigation. The receiving personnel scans the barcode of the Nest tube and looks up the patient information and consent status that is displayed on the computer screen by the Laboratory Information Management System (LIMS). Tubes with complete patient information in the LIMS and no leaks (i.e., eligible samples) are placed in a rack. The location of the sample in the rack should match the assigned location in the LIMS. Unqualified samples are placed in a separate rack and set aside for further investigation by the receiving supervisor. The rack of samples may then be placed in a platform locker in a holding position at 600 rpm until a Medical Laboratory Scientist (MLS) from the sample preparation team retrieves the samples.

[0095] (Combined Swab Preparation Buffer) As part of the sample preparation, the swab sample is mixed with a unique buffer composition (referred to herein as Swab Prep Buffer) specifically formulated for the swab sample. Preparation of this Swab Prep Buffer involves the use of at least the following equipment: a safety cabinet or clean bench (a work area capable of maintaining a sterile environment); sterile, individually wrapped pipettes, pipette tips (e.g., 10 mL and 25 mL); pipette aid; pipettors (1 mL or 200 μL) and corresponding tips; 50 mL sterile, nuclease-free Falcon tubes; Eppendorf repeater (50 mL capacity); 1.9 ml Cryovial tubes (Nest); Nest tube rack; and a screw cap tube decapper device (Brooks Life Sciences).

[0096] The preparation of this swab transport buffer further includes the use of at least the following reagents / components: · 10× TBE buffer (Tris-borate-EDTA, pH 8.2-8.4), sterile, DNase-free, RNase-free, and protease-free grade, Fisher BioReagents, catalog number BP133320, 20 L; ·RNase inhibitor, human placenta, 40,000 units / ml, sterile, DNase-free ·RNase-free grade, New England Biolabs, Catalog No. M0307L, 10,000 units, 250 μl / tube; · Amphotericin B solution, 250 μg / ml in deionized water, sterile, Sigma-Aldrich, Cat. No. A2942, 100 ml (or a similar antifungal agent at an appropriate concentration to prevent fungal contamination and growth); · Penicillin-Streptomycin Solution, 100×, a 100-fold working concentration mixture of penicillin (10,000 IU) and streptomycin (10,000 μg / ml), sterile, Corning, catalog number 30-002-CI (or a similar antibiotic at an appropriate concentration to prevent bacterial contamination and growth); Nuclease-free water, sterile, Millipore / Sigma, W4502, DNase-free, RNase-free and protease-free grade; and Disinfectant (e.g. 70% ethanol).

[0097] Preparation of these components includes at least the following steps: cleaning the work surface with an appropriate disinfectant; sterilizing reagent bottles before placing them on the work surface; dispensing 500 ml / bottle of 10x TBE buffer into sterile 500 ml Corning bottles and storing at room temperature; dispensing 894.95 ml / bottle of nuclease-free water into sterile 1 L Corning bottles and storing at room temperature; dispensing 4 ml / tube of Amphotericin B solution (into sterile 5 ml Corning tubes) and storing at -20°C; dispensing 1 ml / tube of Penicillin / Streptomycin (into sterile Eppendorf tubes) and storing at -20°C; recording lot information and preparation in a laboratory maintained notebook.

[0098] The preparation of this swab preparation buffer includes at least the following steps: 1. Clean the work surface with an appropriate disinfectant; 2. Sterilize reagent bottles before placing them on the work surface (dispensing everything except RNase inhibitor) 3. For example, to prepare 1IL of viral transport buffer, 3.1. Bring one bottle of nuclease-free water (894.95ml / bottle); 3.2. Using a sterile 50ml Falcon tube, add 100ml of 10x TBE buffer; 3.3. Using a sterile pipette, add 50 μl of RNase inhibitor; 3.4. Thaw one tube of amphotericin B solution and one tube of penicillin / streptomycin and use a sterile pipette to aseptically add 4 ml of amphotericin and 1 ml of penicillin / streptomycin to the bottles. 4. Record lot information and preparation in a laboratory maintained notebook; 5. Assign appropriate laboratory identification (e.g., lot number); 6. Cap the tube tightly and mix thoroughly by inverting the tube; 7. Withdraw 100 μl of medium for quality control (QC) sample; 8. In that bottle Swab transport buffer Laboratory ID: (insert appropriate ID for the laboratory (e.g., STB2 for Summit Buffer 2)) DOM: (insert current manufacturing date) Expiration Date: (insert the date one month after the manufacturing date) Store at 2℃~8℃ and label it; 9. Store at 2°C to 8°C until distribution into aliquots; 10. Using an Eppendorf repeater (50 mL capacity) and a Brooks decapper, dispense 1 mL of prepared swab preparation buffer into individual sterile 1.9 mL screw-cap tubes (Nest); 11.Perform sterility checks; 12. Store the tube and any remaining buffer in the bottle at 2°C to 8°C.

[0099] Preparation of combined swab samples A Medical Laboratory Scientist (MLS) from the sample preparation team retrieves a rack of incoming samples from the locker and brings the rack to the sample preparation room to prepare them for testing. The MLS brings a prepared 96-well sample preparation plate (SPP) containing 5 μL / well of protease (proteinase K). Specifically, the 96-well SPP contains 5 μL per well of proteinase K (Promega) dispensed into each well using a multichannel equalizer or Viaflow (Integra). The caps of the samples are decapped using a semi-automated 6-channel decapper (Brooks) or an automated 48 format decapper (Brooks) inside a safety cabinet. If the 6-channel decapper is used, the caps are temporarily placed in its cap carrier rack. Using the E1-ClipTip electronic multichannel (8 channel) equalizer, gently pipette approximately 35 μL of swab sample from the tube in the 48-well rack into a 96-well SPP containing 5 μL of Proteinase K. Two 48-well racks of samples will fill one 96-well SPP. Recap the samples (6 at a time if using a 6-channel decapper, or 48 at a time if using an automated 48 format decapper). Mix the swab samples and Proteinase K thoroughly by placing the plate on a digital microplate shaker at 500 RPM for 1 minute. Place the plate in a miniAmp 96-well PCR instrument at 95°C for 5 minutes and hold at 4°C. Then transport the entire rack of samples to the temporary sample storage area. Any samples requiring repeat testing are identified from this temporary sample storage area. Only one repeat is allowed. In case of failure, request a new sample. Any remaining samples are stored at -80°C for future use.

[0100] (Preparation of PCR reagents and plate configuration (combined swab testing)) The plate containing the PCR master mix (herein referred to as the PCR master mix plate (PMMP)) contains 12.5 μL of PCR master mix dispensed into each well of a 96-well or 384-well plate using a multichannel equalizer or Viaflow (Integra). The PCR master mix is ​​composed of 10 μL of Luna Universal Probe One-Step Reaction Mix, 1 μL of Luna Warmstart RT enzyme mix, and 1.5 μL of Nl / RNP primer / probe. The 1.5 μL of Nl / RNP primer / probe is made by adding 50.25 μL of each 100 μM primer and probe stock to 524 μL of IDTE buffer (pH 7.5) to make a 6.7 μM working stock of Nl primer and a 6.7 μM working stock of RNP primer, and 1.7 μM of FAM-labeled Nl probe and ATTO-647-labeled RNP probe.

[0101] The molecular team Medical Laboratory Scientist (MLS) places a 96-well or 384-well PCR Master Mix Plate (PMMP) into their individual PCR workstation and adds 7.5 μL of the processed combined swab samples from the swab sample preparation step above to each designated well of the PMMP. The processed combined swab samples are then mixed with the PCR Master Mix above by pipetting, taking care to avoid creating bubbles. The MLS then adds 7.5 μL of the positive control (IDT synthetic 2019-SARS-CoV-N control, 4000 copies / μL) and negative control (IDT Hs-RPP30 control, 4000 copies / μL) for SARS-CoV-2, as well as a no template control (NTC-water) to the PCR wells designated for those controls (one positive control, one negative control, and NTC per plate) and mixes by pipetting, avoiding creating bubbles. The MLS then places a clear plastic qPCR film over the PMMP, seals the film with a plate sealer, and centrifuges briefly in a plate centrifuge to remove any bubbles.

[0102] (PCR temperature profile (amplification region) (combined swab test)) Place the plate in a Bio-Rad CFX or QuantStudio PCR instrument, open the master file "ST-COV-PCR protocol" and run the following thermocycling conditions: 1. Step 1: 55°C for 10 min, 1 cycle; 2. Step 2: 95°C for 1 min, 1 cycle; and Step 3: 40 cycles of 95° C. for 10 seconds, 60° C. for 30 seconds (plus reading the plate in both the FAM channel for the N1 target and the Cy5 channel for the RNP target).

[0103] (Data Interpretation (BioRad CFX opus 96-well format) (Combined Swab Testing)) Bio-Rad CFX reports Cq values. The Cq value file (csv file) is then exported from the PCR instrument to OvDx LIMS. The interpretation of the Cq values ​​(DETECTED, NOT DETECTED, and INVALID) is exported to OvDx LIMS according to the following criteria: [Table 2]

[0104] If N1 is detected, the result is valid and returns "DETECTED" regardless of the value for RNP. If N1 is not detected and RNP is <35, return a result of "NOT DETECTED". If RNP Cq value >35 and Nl >36, rescue the sample for retesting. After retesting, if the RNP is still >35, the donor should be contacted and another sample should be collected. NaN = Not a Number.

[0105] (Quality Assurance and Batch Release (Combined Swab Testing)) The Laboratory Supervisor tests the controls including: Positive Control (2019-nCoV_N_Control, IDT), which should be positive for the Nl target but negative for the RNP target; Negative Control (Hs_RPP30 Control), which should be negative for the Nl target but positive for the RNP target; and NTC Control, which should be negative for both the Nl and RNP targets. The Laboratory Supervisor also performs spot testing and evaluates the ratio of positive to negative results. The Medical Director releases the batch after further testing and approves the report.

[0106] (Sample placement after PCR test (swab test)) Samples with INVALID results are identified in the temporary sample storage area (Fume Hood 1) above. Replicate testing is performed on these samples, starting from step III (preparation of saliva sample). Samples with validated results are stored at -80°C. PCR plates are moved to the waste area (Fume Hood 2) as a biohazard.

[0107] Example 3 In this example, the relative effectiveness of anterior nasal swab (ANS) samples and saliva samples was compared for detection of SARS-CoV-2 virus. Briefly, ANS samples were collected with DNA Genotek's OR-100 device (SwabClear™) and saliva samples were collected from the same patients using DNA Genotek's OM-505 device (SalivaClear™). Samples were sufficient to detect SARS-CoV-2 virus according to the manufacturer's instructions.

[0108] While most paired samples showed concordant results between ANS and saliva samples (detection in both or non-detection in both), discordant results between the two specimen types were observed in some paired samples (i.e., SARS-CoV2 detection in one specimen but non-detection in the other). Based on these clinical findings, we hypothesized that the abundance or clearance of SARS-CoV-2 or other respiratory viruses may vary between individuals or at different times during the course of the infection or disease, comparing nasal passages with saliva. Thus, tests that rely on only one specimen site may mean that some SARS-CoV-2 positive cases will be missed.

[0109] Consequently, a test that combines nasal swab and saliva specimens maximizes the chances of detecting SARS-CoV-2 or other respiratory viruses in diverse populations and at different times during the course of the infection or disease.

[0110] Example 4 This example provides experimental results showing improved concentrated viral abundance detected in combined saliva and anterior nostril nasal swab samples compared to paired saliva-only samples.

[0111] Sixteen human participants spat saliva samples into 50 ml Falcon tubes. Anterior nares swab (ANS) samples were collected from the same participants using flocked nasopharyngeal swabs. One saliva sample from each patient was used in an RNA extraction-free qPCR protocol according to Examples 1-2 to detect SARS-CoV-2 infection. The nasal swabs were placed swab-side down into Falcon tubes holding a second saliva sample from each participant. The swabs were squeezed to extract the ANS sample, which was mixed with the saliva. The combined saliva and ANS samples were subjected to the same RNA extraction-free qPCR protocol as the saliva samples above.

[0112] Figure 3 presents the qPCR results as cycle threshold (Ct) values, which indicate how much SARS-CoV-2 virus was detected in the sample. The paired results are presented as "SalivaFast" for the saliva-only samples and "Spit-N-Dip" for the combined samples.

[0113] The data show a significant improvement in concentrated viral load (demonstrated as lower Ct values) in the combined ANS-saliva specimens when compared to saliva-only testing using the same testing protocol.

[0114] Thus, the combined samples clearly provide more sensitive results when compared to samples obtained from a single source. Therefore, combining other types of nasal swabs (e.g., nasopharyngeal and middle turbinate) and mixing such specimens with saliva is expected to produce similar improved results among individuals who may have different viral load levels for detection purposes in separate specimen locations. Similarly, it is also possible to combine nasal and oropharyngeal swabs as a mixed specimen. Thus, while the test provided in this example focuses on combining ANS and saliva as a specimen for testing SARS-CoV-2 and other respiratory viruses, this general method of mixing specimens from different locations in the human body reduces initial testing costs while maximizing the chance of virus detection. This method can also be considered a "pooling" technique that combines specimens from different body locations of the same person.

[0115] (Incorporated by reference) References and citations to other documents (e.g., patents, patent applications, patent publications, journals, books, articles, web content) have been made throughout this disclosure. All such documents are hereby incorporated by reference in their entirety for all purposes.

[0116] (Equivalent) Various modifications of the present invention and many further embodiments thereof, in addition to those shown and described herein, will become apparent to those skilled in the art from the entire contents of this specification, including the references to the scientific and patent literature cited herein. The subject matter herein contains important information, exemplification and guidance that can be adapted to the practice of this invention in its various embodiments and equivalents thereof.

Claims

1. A method for the extraction-free analysis of nucleic acids, comprising: mixing a combined saliva sample and a respiratory mucosa sample in a buffer composition comprising nuclease-free water, an antifungal agent, an antibiotic, and a ribonuclease inhibitor; directly amplifying nucleic acids from the sample in the buffer with primers specific for the target nucleic acid without performing prior extraction of the nucleic acids; and analyzing the amplicons generated in the amplifying step to detect the presence of a pathogen .

2. The method according to claim 1, wherein the nucleic acid is a pathogen.

3. The method according to claim 2, wherein the pathogen is a virus or a bacterium.

4. The method according to claim 3, wherein the virus comprises a coronavirus or an influenza virus.

5. The method according to claim 4, wherein the coronavirus is severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).

6. The method according to claim 5, further comprising analyzing the amplicons to detect the presence of one or more target SARS-CoV-2 variants.

7. The method according to claim 6, which does not include the amplification of wild-type SARS-CoV-2 nucleic acid.

8. The method according to claim 4, wherein the nucleic acid-specific primers target one or more of the N gene, ORF1ab gene, and E gene of the virus.

9. Before the mixing step, placing a nasal swab containing the respiratory mucosa sample into the saliva sample in a container .

10. The method according to claim 9, further comprising squeezing and / or agitating the nasal swab in the sample container to mix the saliva sample and the respiratory mucosa sample.

11. The method according to claim 9, wherein the nasal swab contains a respiratory mucosa sample from one or more locations in the nasal cavity.

12. The method according to claim 11, wherein the one or more locations include the external nares, the middle turbinate, and / or the nasopharynx.

13. The method according to claim 12, wherein the respiratory mucosa sample is obtained from the external nares.

14. The method according to claim 1, wherein the nucleic acid is RNA or DNA.

15. The method according to claim 1, wherein the analyzing step includes sequencing the amplicons.

16. ​ The method according to claim 1, wherein the buffer composition contains a reducing agent.

17. The method according to claim 14, wherein the buffer composition contains a reducing agent which is a tris(2-carboxyethyl)phosphine hydrochloride solution.

18. The method according to claim 1, wherein the antifungal agent contains amphotericin B and the antibiotic contains penicillin-streptomycin.

19. The method according to claim 1, further comprising the step of comparing the amounts of nucleic acids in a plurality of combined saliva samples and respiratory mucosa samples obtained from a patient at consecutive time points, wherein an increase or decrease in the amount of the nucleic acid over time is an indicator of the progression of the disease.

20. The method according to claim 19, wherein the amount of the nucleic acid is an indicator of the outcome of the disease.

21. A method for non-extraction analysis of nucleic acids, comprising: providing a vial; mixing a saliva sample and a respiratory mucosa swab sample obtained from a subject in the vial with a buffer composition containing nuclease-free water, an antifungal agent, an antibiotic, and a ribonuclease inhibitor; directly amplifying nucleic acids from the sample in the buffer with primers specific to the target nucleic acid without performing prior extraction of the nucleic acid; and analyzing the amplicons generated in the amplifying step to detect the presence of a pathogen The method comprising.

22. The method according to claim 21, wherein the saliva sample is obtained from the subject using a saliva collection aid (SCA) or a funnel.

23. The method according to claim 22, wherein the saliva sample is obtained using an SCA containing the buffer composition, and the buffer composition is released into the vial.

24. The method according to claim 22, wherein the SCA or the funnel is provided with a lid.

25. The method according to claim 24, wherein the lid contains the buffer composition, and the buffer composition is released into the vial when the lid is closed.

26. The method according to claim 22, wherein the SCA or the funnel is integrated with the vial.

27. The method according to claim 21, wherein the respiratory mucosa swab sample is obtained by swabbing the outer nostrils of the subject.

28. The method according to claim 21, wherein the swab used to obtain the respiratory mucosa sample is attached to a cap used to seal the vial.

29. The method according to claim 28, wherein sealing the vial with the cap places the swap in the saliva sample.

30. The vial; A saliva collection aid or funnel; The buffer composition; The primer specific for the target nucleic acid; and Instructions for use A kit for carrying out the method according to claim 21, comprising:

31. A method for stabilizing a virus sample, comprising: Adding a buffer containing nuclease-free water, an antifungal agent, an antibiotic, and a ribonuclease inhibitor to a body fluid sample suspected of containing a virus, thereby stabilizing the virus; and Transporting the sample to a testing site A method comprising:

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