Devices and methods for STI pathogen testing without nucleic acid extraction

Abstract

The present invention provides compositions, devices, methods and kits that allow for rapid diagnosis of diseases and pathogens (including sexually transmitted diseases) by direct PCR techniques without nucleic acid extraction. The present invention provides compositions and methods for rapid, extraction-free detection and analysis of nucleic acids in biological samples. More specifically, the present invention provides compositions for processing biological samples to provide nucleic acids available for subsequent amplification and / or detection (e.g., using next-generation sequencing techniques), while eliminating the need for an initial nucleic acid extraction step.

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, especially for sexually transmitted diseases. [Background technology]

[0002] (background) The United States Center for Disease Control estimates that one in five people has a sexually transmitted infection (STI), with over 25 million new cases of STIs each year. As a result, an estimated $16 billion is paid annually in direct medical costs attributable to STIs in the United States. These costs are likely to increase as antibiotic-resistant strains of bacterial STIs become more prevalent and other STIs emerge, with monkeypox emerging in 2022 for example.

[0003] STIs can be caused by a myriad of viruses, bacteria, and microorganisms. The most common STI is Chlamydia trachomatis (CT) infection, with over 1.5 million new cases annually in the United States. Although roughly half as prevalent, Neisseria gonorrhoeae (NG) infection is becoming an increasing concern with the emergence of strains resistant to ceftriaxone, the first-line treatment for NG infections.

[0004] Whether or not an STI is treatable, early detection and surveillance remain important components in reducing the societal burden and prevalence of STIs.

[0005] Current detection techniques for many infectious diseases include 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)) have been developed around the world to detect many viral and bacterial infections.

[0006] However, although current PCR methods allow for 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 their testing protocols. This initial nucleic acid isolation and purification step (i.e., extraction step), which is required in conventional methods before undergoing PCR, constitutes a major obstacle in this diagnostic process. This is because it remains manual, laborious and expensive, further increasing the chances of accidental contamination and human error. Furthermore, the effectiveness of PCR-based tests for diagnosing infectious diseases, particularly sexually transmitted diseases, varies based on the type of sample analyzed (e.g., genital swab, urine, blood, or saliva), the timing of sample collection relative to the course of the infectious disease, and even the subject's behavior prior to sample collection. Summary of the Invention [Means for solving the problem]

[0007] (overview) The present invention provides compositions and methods for rapid, extraction-free detection and analysis of nucleic acids in biological samples. More specifically, the present invention provides compositions for processing biological samples to provide available nucleic acids for subsequent amplification and / or detection (e.g., using next-generation sequencing technology), while eliminating the need for an initial nucleic acid extraction step. Furthermore, the compositions of the present invention eliminate the need for any pathogen transport medium, which is known to inhibit subsequent PCR assays. The compositions of the present invention include, for example, unique buffer compositions for sample transport and preparation, which, when mixed with a sample of interest, can stabilize nucleic acids and prepare the nucleic acids from the sample for direct nucleic acid amplification and analysis without the need for initial nucleic acid extraction (i.e., nucleic acid isolation and purification).

[0008] Advantageously, the method of the present invention may use a non-invasive sample type (e.g., a swab taken from a potentially infected area). The buffers used in the extraction-free method of the present invention stabilize and preserve the target nucleic acid (e.g., from one or more sexually transmitted pathogens) from the non-invasive sample. This allows samples to be collected at home and sent to a laboratory for analysis, or even analyzed at home with a suitable point-of-care testing device. This may be important for adoption of the testing modality by users, especially when testing for sexually transmitted infections (STIs). Self-collection of samples for STIs has been shown to be readily accepted, especially in men. See Yared N et al., Optimizing Screening for Sexually Transmitted Infections in Men Using Self-Collected Swabs: A Systematic Review, Sex Transm Dis., May 2018; 45(5): 294-300, which is incorporated herein by reference.

[0009] In the method of the present invention, sample testing is directly from the sample without nucleic acid extraction step.Instead, the unique buffer composition described herein provides the clinical sample, and the nucleic acid from the sample can be directly used in downstream assays, including downstream diagnostic tests based on qPCR, rtPCR, and / or NGS.The present invention is useful for detecting DNA or RNA, as needed, for detecting one or more sexually transmitted pathogens.Therefore, in a preferred embodiment, the target nucleic acid for detection comprises the nucleic acid sequence associated with one or more sexually transmitted pathogens.

[0010] The method of the present invention is applicable to the detection of any pathogen that can undergo PCR amplification, including viruses (e.g., human papillomavirus (HPV) and monkeypox virus (MPV)), bacteria (e.g., Chlamydia trachomatis (CT) and Neisseria gonorrhoeae (NG)), and other pathogens (e.g., Candida albicans yeast). In certain embodiments, the method of the present invention can detect multiple sexually transmitted pathogens from a single sample, including combined samples. Furthermore, the method of the present invention is suitable for detecting tumor viruses, some of which are sexually transmitted (e.g., HPV). By extension, the method of the present invention can detect one or more types of cancer (e.g., lung cancer, head and neck cancer, cervical cancer).

[0011] Thus, in certain embodiments, the method of the present invention may include detecting one or more genetic markers that are correlated with increased risk of cancer.Such genetic markers may be genetic markers that are correlated with specific pathogens or pathogen variants, for example, genetic markers that are used to distinguish high-risk variants of HPV, including HPV-6, HPV-11, HPV-16, HPV-18, HPV-31, HPV-33, HPV-35, HPV-39, HPV-45, HPV-51, HPV-52 or HPV-68 (see American Cancer Society, Human Papilloma Virus (HPV), Cancer, HPV Testing, and HPV Vaccines: Frequently Asked Questions (October 22, 2013)).Similarly, genetic markers that are correlated with increased risk of cancer may include cancer gene sequences and / or gene mutation sequences (for example, KRAS G12C mutant NSCLC). Exemplary genetic markers include, for example, genetic markers associated with cervical cancer, such as SC6; SIX1; human cervical carcinoma 2 proto-oncogene (HCCR-2); p27; viral oncogene E6; viral oncogene E7; pl6INK4A; Mcm proteins (e.g., Mcm5); Cdc proteins; topoisomerase 2α; PCNA; Ki-67; cyclin E; p-53; PAI1; DAP kinase; ESRI; APC; TIMP-3; RAR-β; CALCA; TSLC1; TIMP-2; DcRl; CUDR; DcR2; BRCA1; p15; MSH2; Rassf1A; MLH1; MGMT; SOX1; PAX1; LMX1A; NKX6-1; WT1; ONECUT1; SPAG9; and Rb (retinoblastoma) protein.

[0012] A preferred method of the present invention is used to detect one or more sexually transmitted infections (STIs) from a minimally invasive sample obtained from a subject. For example, in a preferred embodiment, the sample used in the method of the present invention is obtained as a mucosal swab. Such samples may include one or more of vaginal swabs, cervical swabs, urethral swabs, genital swabs, oral swabs, pharyngeal swabs, nasal swabs, eye swabs, and any combination thereof. In a particular embodiment, the method of the present invention uses a fluid sample from a subject, and the fluid sample may include one or more of urine, vaginal mucosa, saliva, blood, nasal mucosa, sputum, cerebrospinal fluid, pus, nipple aspirate, peritoneal fluid, lymph, sweat, tears, and any combination thereof. In a particular embodiment, a combined swab and fluid sample is used in the method of the present invention to detect a sexually transmitted infection.

[0013] Preferably, the sample includes one or more non-invasive mucosal swabs and / or fluid samples (e.g., urine and / or saliva). Non-invasive sample collection allows patients to collect samples at home or at remote clinics without the need for on-site access to sophisticated laboratory equipment and staff. Advantageously, the extraction-free method of the present invention uses a proprietary buffer composition that allows the target nucleic acid from the sample to be preserved and secured for transport to a laboratory for analysis. Fortunately, the extraction-free method of the present invention and its use of a proprietary buffer composition also allow the target nucleic acid from the sample to be analyzed at home with a suitable point-of-care testing device.

[0014] Since the method of the present invention allows for home sample collection for STI testing, it offers several advantages over traditional in-clinic testing, including privacy. Thus, the method of the present invention may help people who are hesitant to go for in-person testing due to perceived stigma. Furthermore, even if not offered at home, the method of the present invention can be used in fairly simple locations, and the samples can be collected by minimally trained staff. This finds clear utility over centralized locations (e.g., hospitals with specialized staff) in extending STI testing to underserved communities.

[0015] In one embodiment, the present invention allows for the combination of two or more different sample types in a single assay, thereby allowing for more accurate results, especially when testing for multiple (potential) sexually transmitted pathogens. In certain embodiments, the two or more different sample types include two or more different types of mucosal swabs. In certain embodiments, the two or more different sample types include two or more different types of fluid samples. In certain embodiments, the two or more different sample types include one or more mucosal swabs (e.g., vaginal swabs) and one or more fluid samples (e.g., saliva or urine).

[0016] Different STIs (including those that are differentially represented in the population) can provide different quality and / or quantity of target nucleic acid depending on the type of sample.For example, a patient may have multiple STIs, and each STI can only be detected at a specific location in the subject's body (e.g., CT infections are reliably detected from genital swabs, and latent HPV infections are detected from oral swabs).Using the method of the present invention, these different samples can be combined in the buffer solution disclosed herein for transport to the laboratory for further analysis.

[0017] The methods of the present invention can be used to detect any sexually transmitted disease.

[0018] Exemplary STIs detected by the method of the present invention include bacterial vaginosis, CT, cystitis, NG, hepatitis A, hepatitis B, hepatitis C, herpes (herpes simplex type 1 and herpes simplex type 2), HIV, HPV, MPV, lymphogranuloma venereum, molluscum contagiosum, nongonococcal urethritis, pelvic peritonitis, phthirus pubis, syphilis, trichomoniasis, and vaginitis. In certain embodiments, the method of the present invention detects multiple sexually transmitted diseases from a single sample. In preferred embodiments, the method of the present invention detects CT and / or NG infections in a sample. The method of the present invention can be used to detect tumor viruses. Exemplary tumor viruses detected by the method of the present invention include HPV, Epstein-Barr virus (EBV), and hepatitis C virus (HCV).

[0019] In another aspect, the present invention provides a stabilizing buffer that preserves the nucleic acid of one or more sexually transmitted pathogens in a sample. The buffer (described below) stabilizes the nucleic acid of viruses, bacteria, and other pathogens for transport prior to detection of the pathogen nucleic acid. In a preferred embodiment, the transport buffer described herein is added to a liquid sample suspected of containing a pathogen. The sample is then transported to a laboratory for extraction and testing. Because the buffer composition disclosed herein preserves the target nucleic acid, multiple pathogen detection assays can be performed on a single sample and / or sample types can be combined for multiplex pathogen analysis.

[0020] In a first aspect, the present invention provides compositions for processing samples (including combined samples as described herein) to provide nucleic acids available for subsequent amplification and / or detection (e.g., using next generation sequencing techniques), while eliminating the need for an initial nucleic acid extraction step. The compositions of the present invention eliminate the need for pathogen transport media, which typically inhibit PCR.

[0021] The compositions of the invention include, for example, unique buffers for sample transport and preparation which, when mixed with a sample of interest, allow preparation of nucleic acids therefrom suitable for direct nucleic acid amplification and analysis without the need for initial nucleic acid extraction (i.e., nucleic acid isolation and purification).

[0022] In certain embodiments, the present invention includes a kit with all the components necessary to obtain a combined sample, which may preferably be one or more mucosal swabs or urine samples. 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.

[0023] For purposes of the present invention, a target nucleic acid can be a human genome sequence, a human transcript sequence, a cancer gene sequence, a genetic mutation sequence (eg, KRAS G12C mutant NSCLC), a pathogen sequence, or a parasite sequence.

[0024] A preferred method further comprises the step of mixing the sample with an inventive buffer composition that allows for the preparation of nucleic acids suitable for nucleic acid amplification from the biological sample 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 (isolation and purification) steps.

[0025] 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 embodiments, the buffer compositions also function as a transport medium, in which the sample (including any sample collection swabs) is placed directly into a suitable collection vessel containing the buffer composition.

[0026] The method further includes performing one or more PCR assays on the prepared nucleic acid to detect one or more target pathogen nucleic acids. If the target nucleic acids are detected, the patient may be diagnosed as having an STI.

[0027] The step of performing the PCR assay comprises using a target nucleic acid specific primer-probe set. In certain methods, the target nucleic acid specific primer-probes are specific to target nucleic acids of various pathogens in a single sample. In some embodiments, the step of performing the PCR assay comprises using a primer-probe set specific to ribonuclease P (RNP). The extraction method disclosed herein is also useful for detecting human genomic sequences or human RNA sequences because it is agnostic to the source of nucleic acid.

[0028] In certain embodiments, the method of the present invention further comprises quantifying the pathogen 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 a sexually transmitted pathogen or not infected with a sexually transmitted pathogen, the method may further comprise determining the severity of the infection based on the pathogen nucleic acid amount. In some embodiments, the method may further comprise comparing the pathogen nucleic acid amount in multiple biological samples obtained from the patient at sequential time points and determining disease progression based on the increase or decrease in the amount of the nucleic acid over time. The method of the present invention may further comprise predicting disease outcome based on the identity or amount of target nucleic acid in the sample. The method of the present invention may also be used to inform a course of treatment or diagnosis. For example, the results may be used to determine appropriate therapeutic or clinical procedures.

[0029] In another embodiment, the present invention provides detection of bacteria using a buffer without extraction to preserve bacterial DNA and / or RNA for detection. The same buffer is useful for preserving both viruses and bacteria, thereby allowing detection of viral and bacterial pathogens in the same sample or combination of samples. Thus, in one embodiment, the present invention provides a method to stabilize bacteria and / or viruses in a biological sample, for example, for PCR testing without extraction. 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, since the buffer disclosed herein also stabilizes viruses, a single test and sample can also be used to detect viral STIs (e.g., HPV, HIV, MPV, and herpes).

[0030] The present invention also provides a method for analyzing nucleic acid without extraction. An exemplary method includes a mucosal swab sample from a subject. Alternatively, or in addition, the method can include obtaining additional mucosal swabs (e.g., from another body location) and / or one or more fluid samples (e.g., urine). When multiple samples are used, the method of the present invention can include combining the samples in a vial.

[0031] The sample, including 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. The method thus includes a step of directly amplifying the nucleic acid in the buffer with a primer specific for the target nucleic acid. The direct amplification is performed without a prior nucleic acid extraction step. After amplification, the method includes a step of analyzing the amplicons generated in the amplifying step to detect the presence of one or more pathogens.

[0032] In certain embodiments, the sample is a fluid sample. The fluid sample may be obtained using a collection aid. For example, if the fluid sample is saliva, the sample may be obtained from the subject using a sample collection aid or a funnel. The sample collection aid may include the buffer composition, which is released into the vial. For example, the sample collection aid may include the buffer composition in an internal pouch or compartment or in a lid, which releases the buffer composition into the vial. In certain embodiments, the sample collection aid or funnel comprises a lid. The lid may include the buffer composition, which is released into the vial when the lid is closed. In certain embodiments, the sample collection aid or funnel is integrated with the vial. Alternatively, the sample collection aid or funnel may be configured to connect to the vial during saliva collection. In certain embodiments, the sample collection aid or funnel is configured such that it can be reversibly connected to the vial.

[0033] In a preferred embodiment, the sample is obtained using one or more mucosal swabs. In a particular embodiment, the mucosal swabs include one or more of vaginal swabs, cervical swabs, urethral swabs, genital swabs, oral swabs, pharyngeal swabs, nasal swabs, eye swabs, and any combination thereof. In a particular embodiment, the swab used to obtain the mucosal sample is attached to the cap used to seal the vial. Sealing the vial with the cap can place the swab in the fluid sample to form a combined sample. Alternatively, a first swab can be added to the vial and a second swab is added. One of the swabs can be attached to the cap of the vial.

[0034] 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 embodiments, the kits of the present invention comprise one or more vials, sample 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]

[0035] [Figure 1] FIG. 1 shows a schematic diagram of an extraction-free real-time RT-qPCR test for quantitative detection of nucleic acid from one or more sexually transmitted pathogens in mucosal swabs collected and processed with the unique buffer composition of the present invention.

[0036] [Diagram 2] FIG. 2 shows a sample from a patient suspected of having an STI 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.

[0037] [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.

[0038] [Figure 4] FIG. 4 depicts selected components used in the methods of the present disclosure and provided in certain kits of the present invention.

[0039] [Diagram 5] FIG. 5 shows selected components of a kit of the invention for detecting a target nucleic acid in a combined sample.

[0040] [Figure 6] FIG. 6 shows qPCR readouts for assays using the methods of the invention without nucleic acid extraction to detect CT and NG nucleic acids from swab and fluid samples.

[0041] [Figure 7] FIG. 7 summarizes the qPCR assays and resulting data of FIG. 6 for singleplex assays using probes and primers for NG.

[0042] [Figure 8] FIG. 8 summarizes the qPCR assays and resulting data of FIG. 6 for singleplex assays using probes and primers for CT.

[0043] [Figure 9] FIG. 9 shows qPCR readouts for assays using the methods of the invention without nucleic acid extraction to detect CT and NG nucleic acids from swab and fluid samples.

[0044] [Figure 10] FIG. 10 summarizes the qPCR assays and resulting data of FIG. 9 for singleplex assays using probes and primers for NG.

[0045] [Figure 11] FIG. 11 summarizes the qPCR assays and resulting data of FIG. 9 for singleplex assays using probes and primers for CT.

[0046] [Figure 12] FIG. 12 summarizes the qPCR assays and resulting data of FIG. 9 for a multiplex assay using probes and primers for CT and NG.

[0047] [Figure 13]FIG. 13 presents a chart showing consistent results across samples and assays using the methods of the present invention without nucleic acid extraction to detect STIs. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0048] (Detailed Description) The present invention provides compositions, methods, and kits that allow for rapid diagnosis of sexually transmitted diseases by direct PCR techniques without extraction using minimally invasive samples. The present invention also provides buffers that stabilize and preserve target nucleic acids in samples, allowing for extraction-free testing of pathogen nucleic acids (particularly, extraction-free testing of multiple pathogens (e.g., multiple samples from one individual) derived simultaneously from one or more sources). Thus, the methods of the present invention include methods for viral testing, bacterial testing, or combinations. Furthermore, the buffers taught herein preserve samples (e.g., nucleic acids derived from viral STIs (e.g., HPV and HIV), bacterial STIs (e.g., CT and NG), other pathogens, and cancer genes), so that the samples can be transported without substantial loss of their target pathogens and / or nucleic acid sequences.

[0049] The compositions, methods, and kits of the present invention can be used to process biological samples and provide available DNA for subsequent PCR assays while eliminating the need for an initial nucleic acid 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 isolation and purification). Thus, unlike many previous approaches that include a nucleic acid extraction step, the direct sample testing of the present invention simplifies 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 dissolution in the buffer. The inactivated sample can then be used for downstream qPCR diagnostic testing.

[0050] As a result, the compositions, methods, and kits of the present invention improve upon traditional pathogen testing and detection approaches by reducing the number of steps required for sample preparation and testing. In turn, 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 operators from the samples, while reducing labor costs and consumables. The efficiency and cost savings are magnified by testing for multiple STIs using a single sample.

[0051] It should be noted that the method described herein can be used to diagnose various infectious diseases, including microbial, viral and cancer. However, for simplicity and ease of explanation and examples, the following describes a method for diagnosing STIs by direct PCR approach without extraction. It should be noted that STIs can be diagnosed using the method of the present invention that targets nucleic acid from tumor viruses, some of which are sexually transmitted (e.g., HPV). By extension, the method of the present invention can detect one or more types of cancer (e.g., lung cancer, head and neck cancer, cervical cancer).

[0052] The methods of the invention provide rapid detection of STIs (i.e., the presence of sexually transmitted pathogens in patients) by reducing the number of steps in sample preparation that are typically required in conventional STI detection methods that rely on PCR assays. Additionally, the methods of the invention that use combined samples allow tests to be performed simultaneously on samples obtained from locations harboring high concentrations of pathogens, including at various times during the course of an infectious disease.

[0053] Preferably, the samples used in the methods of the present invention include one or more non-invasive mucosal swabs and / or fluid samples (e.g., urine and / or saliva).Non-invasive sample collection allows patients to collect samples at home or in remote clinics without the need for on-site access to sophisticated laboratory equipment and staff.Advantageously, the methods of the present invention without extraction use proprietary buffer compositions that allow target nucleic acids from the sample to be preserved and secured for transport to a laboratory for analysis.

[0054] Fortunately, the extraction-free method of the present invention and use of its proprietary buffer compositions also allow the target nucleic acid from the sample to be analyzed at home with a suitable point-of-care testing device.

[0055] Since the method of the present invention allows for home sample collection for STI testing, it offers several advantages over traditional in-clinic testing, including privacy. Thus, the method of the present invention may help people who are hesitant to go for in-person testing due to perceived stigma. Furthermore, even if not offered at home, the method of the present invention can be used in fairly simple locations, and the samples can be collected by minimally trained staff. This finds clear utility over centralized locations (e.g., hospitals with specialized staff) in extending STI testing to underserved communities.

[0056] In general, the workflow for the exemplary methods of the present invention includes obtaining a biological sample from an individual. The method of sample collection, as well as the type of sample collected, may depend on the particular sexually transmitted disease being tested for. For example, the samples used in the present invention may include one or more mucosal swabs or bodily fluid samples collected in any clinically acceptable manner.

[0057] Mucosal samples may include biological material from one or more of vaginal swabs, cervical swabs, urethral swabs, genital swabs, oral swabs, pharyngeal swabs, nasal swabs, eye swabs, and any combination thereof. Bodily fluid samples may be liquid material, for example, from humans or other mammals. Such bodily fluids include, but are not limited to, mucus, blood, plasma, serum, serum derivatives, bile, maternal blood, phlegm, saliva, sputum, 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). Samples may also include media containing cells or biological material. Samples may also include blood clots (e.g., blood clots obtained from whole blood after serum has been removed).

[0058] The swabs used in the methods herein may be squeezed or agitated to extract the sample and, in certain embodiments, mix it with another portion of the combined sample (e.g., saliva). In certain embodiments, a bodily fluid sample is collected and a swab is placed in the sample for sample preparation.

[0059] As mentioned above, many current STI testing approaches rely on an initial step of isolating and purifying nucleic acid from clinical samples. For example, in many previous methods, the application of qPCR for relative quantification of the nucleic acid of interest is preceded by steps that may include: (1) isolation and purification of total nucleic acid from the sample; (2) elution and possible concentration of the material; and / or (3) use of purified RNA in a reverse transcription (RT) reaction that produces complementary DNA (cDNA), which is then utilized for the qPCR reaction. The initial nucleic acid isolation and purification step (i.e., extraction step) required in these methods before undergoing PCR constitutes a major obstacle in the diagnostic process. This is because it remains manual, laborious and expensive, further increasing the chance of accidental contamination and human error.

[0060] The present invention provides a composition for processing samples and providing DNA that can be used for subsequent PCR assays, while eliminating the need for an initial nucleic acid extraction step.For example, a unique buffer composition is used for sample preparation, so 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 isolation and purification).

[0061] 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, 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, can be 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.

[0062] For example, DNA can be synthesized by reverse transcription from viral RNA associated with a 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 more thermostable RT, in which the reaction is carried out at higher temperatures, can be very useful when dealing with RNA that contains a large amount of secondary structure.

[0063] Digital polymerase chain reaction (dPCR) is an improvement of conventional polymerase chain reaction method, which 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 individually, 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.

[0064] 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.

[0065] The compositions and methods of the present invention can be used to detect any pathogen-specific nucleic acid, but in a preferred embodiment, one or more sexually transmitted pathogens are the detection target.The methods of the present invention can be used to detect any sexually transmitted disease.Exemplary STIs detected by the methods of the present invention include bacterial vaginosis, CT, cystitis, NG, hepatitis A, hepatitis B, hepatitis C, herpes (herpes simplex type 1 and herpes simplex type 2), HIV, HPV, MPV, lymphogranuloma venereum, molluscum contagiosum, nongonococcal urethritis, pelvic peritonitis, pubic lice, syphilis, trichomoniasis, and vaginitis.In certain embodiments, the methods of the present invention detect multiple sexually transmitted diseases from a single sample.In a preferred embodiment, the methods of the present invention detect CT infection and / or NG infection in a sample.

[0066] The method of the present invention can be used to detect tumor viruses. Exemplary tumor viruses detected by the method of the present invention include HPV, Epstein-Barr virus (EBV), and Hepatitis C virus (HCV). The method of the present invention is suitable for detecting tumor viruses, some of which are sexually transmitted (e.g., HPV). By extension, the method of the present invention can detect one or more types of cancer (e.g., lung cancer, head and neck cancer, cervical cancer).

[0067] Thus, in certain embodiments, the method of the present invention may include detecting one or more genetic markers that are correlated with increased risk of cancer.Such genetic markers may be genetic markers that are correlated with specific pathogens or pathogen variants, for example, genetic markers that are used to distinguish high-risk variants of HPV, including HPV-6, HPV-11, HPV-16, HPV-18, HPV-31, HPV-33, HPV-35, HPV-39, HPV-45, HPV-51, HPV-52 or HPV-68 (see American Cancer Society, Human Papilloma Virus (HPV), Cancer, HPV Testing, and HPV Vaccines: Frequently Asked Questions (October 22, 2013)).Similarly, genetic markers that are correlated with increased risk of cancer may include cancer gene sequences and / or gene mutation sequences (for example, KRAS G12C mutant NSCLC). Exemplary genetic markers include, for example, genetic markers associated with cervical cancer, such as SC6; SIX1; human cervical carcinoma 2 proto-oncogene (HCCR-2); p27; viral oncogene E6; viral oncogene E7; pl6INK4A; Mcm proteins (e.g., Mcm5); Cdc proteins; topoisomerase 2α; PCNA; Ki-67; cyclin E; p-53; PAI1; DAP kinase; ESRI; APC; TIMP-3; RAR-β; CALCA; TSLC1; TIMP-2; DcRl; CUDR; DcR2; BRCA1; p15; MSH2; Rassf1A; MLH1; MGMT; SOX1; PAX1; LMX1A; NKX6-1; WT1; ONECUT1; SPAG9; and Rb (retinoblastoma) protein.

[0068] In certain embodiments, the methods of the present invention include targeting one or more endogenous nucleic acids (e.g., genomic DNA / RNA or mRNA transcripts) or gene targets of a subject using one or more samples comprising the stabilized buffer composition described herein. Thus, for example, the methods of the present invention may include one or more human genome sequences, human transcript sequences, cancer gene sequences, and / or gene mutation sequences (e.g., KRAS G12C mutant NSCLC) as targets. The methods of the present invention may include evaluating one or more endogenous nucleic acids for mutations indicative of a disease (e.g., cancer) or other condition (e.g., a predisposition to cancer development or progression). Mutations detected using the methods of the present invention may include, for example, somatic mutations (which may be indicative of cancer / tumor or minimal residual disease). In certain embodiments, the methods of the present invention include evaluating the methylation status of one or more target nucleic acids. DNA methylation plays a role in regulating gene expression, and aberrant DNA methylation is associated with many diseases, including cancer. DNA methylation profiling (including longitudinal profiling) is a valuable diagnostic tool for cancer detection, diagnosis, and / or monitoring.For example, specific patterns of differentially methylated regions and / or allele-specific methylation patterns can be useful as molecular markers for non-invasive diagnosis using target nucleic acid obtained using the method of the present invention without nucleic acid extraction.

[0069] In certain embodiments, the target nucleic acid is used to monitor or evaluate the progression of disease or condition (e.g., cancer or infectious disease) in a subject. Evaluating disease according to the method of the present invention can include one or more of predicting disease severity, diagnosing or determining the stage of disease progression, classifying cancer type, and predicting drug response. Certain methods of the present invention can include obtaining target nucleic acid from a sample, which is used to diagnose tumor before the tumor is visible. This allows earlier treatment than that provided by existing diagnostic modalities.

[0070] The method of the present invention can be used to provide longitudinal evaluation of a subject's disease or condition.For example, longitudinal evaluation can include obtaining samples from a subject at multiple time points in time and amplifying target nucleic acid using the method of the present invention without nucleic acid extraction.The amplicons evaluated from multiple time points can be used to evaluate, for example, the progression of cancer, the development of a specific subtype of cancer, minimal residual disease, possible risk of metastasis, any advantage in further monitoring, changes in methylation status or methylation pattern, gene expression pattern, etc.

[0071] The compositions and methods of the present invention for the detection of sexually transmitted diseases include the use of one or more PCR assays (e.g., ddPCR) of target nucleic acids obtained from mucosal swab samples and / or bodily fluid samples. Further, in some embodiments, performing the one or more PCR assays includes using a primer-probe set specific for ribonuclease P (RNP).

[0072] In addition to diagnosing an individual as infected with an STI, the method of the invention may further comprise determining the severity of the infection based on the amount of target nucleic acid in the sample.For example, the method of the invention is useful for assessing viral or bacterial load, which may directly correlate with the severity and / or progression of the disease.In some embodiments, the method may further comprise comparing the amount of target nucleic acid in multiple combined biological samples obtained from the patient at sequential time points, and determining the progression of the disease based on the increase or decrease in the amount of the target nucleic acid over time.The method of the invention may also be used to predict the outcome and / or severity of the disease based on the amount of the target nucleic acid.

[0073] FIG. 1 shows a schematic diagram of a real-time qPCR test without extraction for quantitative detection of nucleic acid from one or more sexually transmitted pathogens in a biological specimen (e.g., a swab sample and / or a bodily fluid sample) collected and treated with the unique buffer composition of the present invention. In certain embodiments, a bodily fluid sample is collected in an acceptable container. A swab, spatula, brush, or similar device is used to collect a mucosal sample and then placed in the container containing the bodily fluid sample. The swab can be squeezed or agitated to extract the mucosal sample and mix it with the bodily fluid sample. 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 embodiments, the buffer composition can be used for sample preparation and / or transport medium.

[0074] After collecting the samples and providing them with the unique buffer composition, viral particles and / or bacteria can be inactivated either by heating or by direct lysis in the buffer. The inactivated samples can then be used for downstream qPCR diagnostic testing without the need for an additional nucleic acid extraction step (isolation and purification) that conventional approaches rely on.

[0075] In certain embodiments, the prepared samples can be transferred to a PCR plate (96-well / 384-well) format where cDNA synthesis by RT (if required) and / or detection by qPCR can be performed.

[0076] FIG. 4 shows certain components used in the methods of the invention. In certain embodiments, 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 embodiments, the vial is provided with a buffer composition 405. The buffer composition is, for example, a pathogen nucleic acid 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.

[0077] Preferably, the vial is at least 1.5 mL so that it can accommodate at least one swab sample and / or bodily fluid sample and the buffer composition of the present invention. 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)).

[0078] In certain embodiments, the vial includes threads 407 or other means for securing a cap, lid, funnel, and / or bodily fluid sample collection aid (shown as a saliva collection aid in FIG. 4). In certain embodiments, threads 407 or other fastening means are used to secure a cap 409 to the vial to seal the sample for transport and / or storage. In certain embodiments, the cap 409 includes a compartment or pouch 411. Securing the cap 409 to the vial pierces or otherwise releases a buffer composition from inside the compartment or pouch 411 into the vial 403.

[0079] In certain embodiments, the methods and kits of the present invention include a means for collecting a bodily fluid sample from a subject. In some methods and kits, the subject simply provides a bodily fluid sample in a sterile vial 403. Alternatively, a sample collection aid 413 (e.g., a saliva collection aid) or a funnel 415 is provided to facilitate collection. The sample collection aid 413 or funnel 415 may include a means (e.g., threads 417) for connecting the collection aid / funnel to the vial during fluid collection. Alternatively, the funnel or collection aid is integrated with the vial to form a single unit.

[0080] Preferably, when provided as a diagnostic kit, the collection aid / funnel is pre-attached to the vial. The collection aid / funnel may include a means for sealing the sample (e.g., a lid or cap). Alternatively, the collection aid / funnel may be removed, for example, by a thread and screw attachment means. Once removed, the collection aid / funnel may be replaced by a cap or lid to seal the sample in the vial.

[0081] The collection aid 413 or funnel 415 may include a pouch or compartment that contains a buffer composition (e.g., a transport buffer as disclosed herein). The pouch or compartment may release the buffer during sample collection. For example, the pouch or compartment may be integrated into a lid or cap for a funnel / collection aid, such as that used in the OME-505 collection kit (DNA Genetek, Inc., Ottawa, Canada). Upon closing the lid or cap, the compartment is pierced, thereby releasing the buffer into the vial containing the sample.

[0082] The methods and kits of the invention also include or use a swab for collecting one or more mucosal samples. In certain embodiments, 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 buffer (and any fluid sample) in the vial is sufficient to cover the swab. However, the level 425 of the fluid sample / buffer does not need to cover the swab. Rather, it is only necessary that the fluid sample / buffer be in a sufficient amount that they and the swab can be mixed in the vial.

[0083] 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 located within a buffer solution in the vial.

[0084] In a preferred embodiment, 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 the sample at home.By adding a pre-measured novel transport buffer composition of the present invention to the 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.

[0085] FIG. 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 sexually transmitted pathogen). The kit includes instructions, including the steps necessary to obtain a sample (e.g., a swab and / or a bodily fluid sample). The instructions outline that a vial 503 is provided to the subject along with a sample collection tool (e.g., a swab, brush, spatula, paddle, or similar tool for obtaining a mucosal swab, and / or a tool such as a fluid sample collection aid 505. As shown, in certain kits of the invention, the vial 503 is pre-filled with a transport buffer 509 as described herein.

[0086] In the kit shown, the subject provides a sample (e.g., urine or saliva) to the vial using a provided bodily fluid sample collection aid 505. As shown, the fluid sample collection aid 505 is shaped to fit snugly into the opening of the vial 503 to facilitate sample collection.

[0087] The kit also includes a swab 511, which is used to obtain a mucosal swab (e.g., a vaginal swab). The handle of the swab includes a break point 513. After the swab is used to obtain a sample, the handle is broken off at the break point. The shortened swab is placed into the vial containing the bodily fluid sample and buffer. The vial is then sealed with a cap for storage or transport. In certain embodiments, the kit includes materials for the subject to mail the combined sample to a laboratory for analysis.

[0088] While the kit shown in FIG. 5 includes provisions for obtaining a bodily fluid sample as described herein, the present invention contemplates kits that do not require a bodily fluid sample, kits that require multiple bodily fluid samples, and kits that require multiple mucosal swabs that may be combined in certain methods of the present invention.

[0089] In certain embodiments, the kit comprises one or more primers, at least one of which is used for the amplification and / or detection of a target nucleic acid in a sample.

[0090] 2 illustrates a mucosal sample 102 for STI testing collected from a patient and loading the sample into an instrument 200 that can perform one or more assays on the sample to determine whether one or more target nucleic acids associated with at least one sexually transmitted pathogen are present in the sample. As described in more detail herein, the sample 102 obtained (12) from the patient can be contained in a suitable container 104. In certain embodiments, the patient is suspected of having one or more STIs, for example, by exhibiting symptoms or due to reports of sexual contact with a person suspected of having an STI. Alternatively, the methods of the present invention can be used for ongoing patient monitoring and / or for periodic STI testing.

[0091] Samples may be collected and stored in a sample-specific container (e.g., centrifuge tube, e.g., screw-cap cryovial). Preferably, 1.9 ml screw-capped cryovials are used. A swab or similar tool with a proximal breakpoint is used, allowing the swab to be inserted into the tube after sample collection. The screw cap is important to prevent contamination. Standard size cryovials allow direct sample storage without additional sample transfer. If desired, a funnel or sample collection aid may be used to facilitate collection of bodily fluid samples.

[0092] 2 further illustrates the loading of 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 one or more target nucleic acids associated with at least one sexually transmitted pathogen are present in the sample. 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 target DNA and / or target RNA, reverse transcription of any target RNA to generate cDNA, amplification of target DNA / target cDNA (operation 16), analysis of data from the amplification step (operation 18), and generation of a report 300 (operation 20) providing information relevant to the STI evaluation.

[0093] 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 (including whether the sample tested positive or negative for one or more target pathogens), and if positive, further metrics (including disease progression and predicted disease outcome). EXAMPLES

[0094] (Example) The following examples provide exemplary protocols for detecting target pathogen nucleic acid according to the methods of the present invention. The following examples show that, among other aspects of the present invention, the methods can successfully provide DNA available for pathogen testing without a nucleic acid extraction step. Furthermore, as shown, the methods of the present invention are applicable to samples obtained by mucosal swabs, body fluid samples, and combined swabs and body fluid samples. Furthermore, the methods of the present invention can detect both viruses and bacteria from samples, and the methods of the present invention can also detect and distinguish multiple sexually transmitted pathogens using a single test.

[0095] Example 1 - Extraction-free methods for swabs, body fluids, and combined samples PCR without extraction relies in part on the effectiveness of proteinase K (PK) digestion, which would otherwise degrade the desired DNA or RNA sample. Various buffer components were tested to optimize for PK activity in either the swab or saliva matrix. This is particularly important for swab samples. Unlike bodily fluid samples (e.g., urine and saliva), which can be collected and transported as raw samples, swab samples should be stored in a transport medium (e.g., viral and / or bacterial transport medium). However, for many sexually transmitted pathogens, traditional swab samples in transport usually require a nucleic acid (e.g., DNA or RNA) extraction step for testing.

[0096] We tested various buffer components, viral transport media, and a commercially available swab collection device, OR100 (DNA Genotek), for PCR without extraction. Negative swab samples were collected from healthy volunteers and placed in each solution.

[0097] Samples were then added to heat-inactivated SARS-CoV-2 virus and mixed with PK by dispensing samples into 96-well plates pre-filled with either a mixture of Saliva Preparation Buffer (see below) and PK (Promega) for saliva samples, or PK alone for swab samples. For saliva samples (SalivaFAST), 30 μL from a single saliva sample was mixed with 5 μL of Saliva Preparation Buffer and 5 μL of PK in each well of the plate. For swab samples (SwabFAST), 35 μL from a single swab sample was mixed with 5 μL of PK per well. The prepared sample plate was then placed on a digital microplate shaker at 500 RPM for 1 minute, and then placed in a thermal cycler at 95°C for 5 minutes for heat inactivation.

[0098] Swab samples in PBS, viral transport medium, and OR100 did not produce positive signals in the N1 region. Among the positive signals, the contrived swab samples in Tris-borate-EDTA (TBE) buffer produced the strongest quantification cycle (Cq) value, which contains the buffer components for the viral transport buffer of the present invention. Similarly, various buffer components, raw saliva, and a commercially available saliva collection device, OM505 (DNA Genotek), were tested for PCR without extraction. Contrived saliva samples in OM505 did not produce positive signals in the N1 region. Among the positive signals, contrived saliva samples in Tris(2-carboxyethyl)phosphine (TCEP) buffer condition produced the strongest Cq value. This is used to improve the PK effectiveness in the SalivaFAST protocol.

[0099] Thus, the methods of the present invention, which do not involve nucleic acid extraction using the transport buffers described herein, allow for stable storage and detection of target nucleic acids from both swab and fluid samples.

[0100] We then tested the relative effectiveness of mucosal swab samples and bodily fluid samples. Anterior nasal swab (ANS) samples and saliva samples were 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 run to detect SARS-CoV-2 virus according to the manufacturer's instructions.

[0101] 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., detection of SARS-CoV2 in one specimen but not 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 in the nasal cavity compared to saliva. Thus, a test that relies on only one specimen site may mean that some positive cases will be missed. This is true for the STI detection method of the present invention, since many sexually transmitted pathogens can be detected differently between various sample types (including throughout the course of the infection).

[0102] The inventors further obtained experimental results showing that the method of the present invention can use combined swab and body fluid samples. Sixteen human participants spat saliva samples into 50 ml Falcon tubes. Flocked nasopharyngeal swabs were used to collect anterior nares swab (ANS) samples from the same participants. One saliva sample from each patient was used in a qPCR protocol without RNA extraction to detect SARS-CoV-2 infection. The nasal swabs were placed swab-side down in a Falcon tube 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 qPCR protocol without RNA extraction as the saliva samples above.

[0103] 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. The data show a marked improvement in concentrated viral abundance (shown as lower Ct values) in the combined ANS-saliva specimens when compared to saliva-only testing using the same testing protocol.

[0104] Thus, for certain pathogens and / or sample types, the combined mucosal swab and bodily fluid sample provides more sensitive results when compared to samples obtained from a single source. Thus, the methods disclosed herein may be used to combine one or more mucosal swabs with one or more bodily fluid samples for STI detection testing to maximize the chances of detecting a sexually transmitted pathogen of interest among diverse populations and at various times during the course of an infection or disease.

[0105] Example 2 - Exemplary Protocol for STI Detection Using a Method Without a Nucleic Acid Extraction Step The present disclosure provides this exemplary protocol for a method of performing STI detection for CT and / or NG infections without nucleic acid extraction, however, the method of the present invention may be used to detect nucleic acid from any other sexually transmitted pathogen.

[0106] (Collecting swab samples) The swab collection device includes: a 1.9 ml Nest tube filled with 1 ml of a unique buffer composition specific to the swab sample (hereinafter referred to as Swab Transport Buffer), which serves as a container for the swab sample; and at least one swab, which is used to swab the patient's mucosa and then placed inside the tube filled with the Swab Transport Buffer.

[0107] The swabs may be collected under the supervision of trained health care personnel designated by the agency overseeing the collection site. Alternatively, kits may be sent to patients at home or other remote facilities. The health care personnel overseeing the collection or the patient obtaining the sample should clean their hands with an alcohol-based sanitizer or unscented soap and water. Prior to collection, the patient is presented with instructions. The patient may provide patient information (including name, date of birth, and any additional information required). The health care personnel may ask the patient to review the study consent form (provided by Ovation) to decide to participate or not participate in the study. Finally, the health care personnel scans the pre-printed barcode label, tying it to the patient information already collected, and then places the label on the tube used by the patient for sample collection.

[0108] For collection, remove the cap of the Nest tube and take 10 swabs from the mucosa of interest for the particular test. Break the handle of the swab at the proximal break point inside the tube. Replace the cap of the Nest tube with the swab inside and securely tighten the cap. If any sample is spilled during this collection process, use an alcohol wipe or equivalent to wipe the outside of the tube to prevent contamination. The samples are then placed in individual bags at room temperature before shipping to the laboratory. (Receiving and accessioning samples in the laboratory)

[0109] The swab samples are transported to the laboratory. The samples are removed from the bag and visually inspected by the Accession Supervisor at the receiving desk for any leaks or damage. Samples that pass the pre-screening step by the supervisor are moved to a desk used by the Accession Team. Samples that do not pass the pre-screening step are set aside for further investigation. The Accessioner scans the barcode of the Nest tube and looks up the patient information and consent status shown 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 Accession 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.

[0110] (swab preparation buffer) As part of sample preparation, the swab sample is mixed with a unique buffer composition (herein referred to as Swab Prep Buffer) specifically prepared for the swab sample. Preparation of the Swab Prep Buffer involves the use of at least the following equipment: a safety cabinet or laminar flow hood (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; Eppendorf repeater (50 mL capacity); 1.9 ml Cryovial tubes (Nest); Nest tube rack; and a screw cap tube decapper device (Brooks Life Sciences).

[0111] The preparation of the swab transport buffer further comprises 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).

[0112] 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.

[0113] The preparation of the 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 1 L of 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, using a sterile pipette, 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 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.

[0114] (Sample preparation) The MLS from the sample preparation team retrieves a rack of received samples from the locker and brings it to the sample preparation room to prepare them for testing. The MLS brings a prepared 96-well Sample Prep Plate (SPP) containing 10 μL / well of Sample Prep Mix (SPM). The SPM contains sample preparation buffer and protease (proteinase K). In detail, the 96-well SPP contains 10 μL of SPM per well (5 μL sample preparation buffer and 5 μL proteinase K (Promega)) that is dispensed into each well using a multichannel equalizer or Viaflow (Integra). 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 the cap carrier rack. Pipette approximately 30 μL of sample from the tubes in the 48-well rack into a 96-well SPP containing 10 μL of SPM using the E1-ClipTip electronic multichannel (8-channel) equalizer thoroughly. 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 samples and SPM 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 a temporary sample storage area. Identify any samples that require a repeat from this temporary sample storage area. Only one repeat is allowed. If unsuccessful, request a new sample. Store any remaining samples at -80°C for future use.

[0115] (Preparation of PCR reagents and plate configuration) 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 pathogen-specific / RNP primers / probes. The 1.5 μL of pathogen-specific / RNP primers / probes are generated by adding 50.25 μL of each 100 μM primer and probe stock to 524 μL of IDTE buffer (pH 7.5) as 66.7 μM working stocks of pathogen-specific and RNP primers, and 1.7 μM of FAM-labeled pathogen-specific and ATTO-647-labeled RNP probes. Alternatively, if multiple pathogens are detected in a single sample, 1.5 µL of pathogen-specific / RNP primers / probes are 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 66.7 µM working stocks of pathogen-specific and RNP primers, and 1.7 µM of differently labeled pathogen-specific probes (e.g., Rox-labeled probe for NG detection and Fam-labeled probe for CT detection) and ATTO-647-labeled RNP probe.

[0116] The molecular team's MLS places the 96-well or 384-well PMMPs into their individual PCR workstations and adds 7.5 μL of the processed samples from the sample preparation step above to each designated well of the PMMP. The processed samples are then mixed with the PCR master mix above by pipetting, taking care to avoid creating bubbles. The MLS may add 7.5 μL of the positive controls (e.g., from inactivated CT / NG swabs, such as those from Microbiologies and Seracare) and negative controls, as well as no template controls (NTC-water), to the PCR wells designated for those controls (one positive control, one negative control, and NTC per plate), mixing by pipetting to avoid 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 bubbles.

[0117] (PCR temperature profile (amplified region)) Load the plate into a Bio-Rad CFX or QuantStudio PCR instrument, open the master file, and run the following thermal cycler 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 plate reading in both the FAM channel for CT targets and / or the Rox channel for NG targets and the Cy5 channel for RNP targets).

[0118] (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 2] If CT / NG is detected, the result is valid and returns "DETECTED" regardless of the value for RNP. If CT / NG is not detected and RNP is ≦25, return a result of "NOT DETECTED". If RNP Cq value >25 and CT / NG >25, rescue the sample for retesting. After retesting, if the RNP is still >25, the donor must be contacted to collect another sample. NaN = Not a Number.

[0119] (Quality Assurance and Batch Release) The laboratory supervisor tests controls including: a positive control (which should be positive for the NG / CT target but negative for the RNP target); a negative control (which should be negative for the NG / CT target but positive for the RNP target); and an NTC control (which should be negative for both the NG / CT and RNP targets). The laboratory supervisor also performs spot checks and estimates the ratio of positive to negative results. The medical director releases the batch after further testing and approves the report.

[0120] (Sample placement after PCR testing) Samples with INVALID results are identified in the temporary sample storage area (furnace hood 1) above. Replicate testing is performed on these samples, starting from step III (sample preparation). Samples with verified results are stored at -80°C. PCR plates are removed to the waste area (furnace hood 2) as a biohazard.

[0121] Example 3 - STIFast Addition Experiment Using a protocol similar to that presented above, we developed STIFast, a nucleic acid extraction-free method for detecting sexually transmitted pathogens from samples. To test the ability of STIFast to detect sexually transmitted diseases, we performed a series of spike experiments that demonstrated the ability of the method to detect the presence of either CT or NG in samples.

[0122] In the following proof-of-concept experiments, CT / NG samples were provided on inactivated swabs. The CT / NG swabs included Helix Elite™ CT / NG control swabs made by Microbiologies, Inc. These swabs contained 1×10 CT and NG bacteria. 3 ~5×l0 3 In other studies, their CT / NG swabs contained ACCURUN molecular control by Seracare, which provides CT / NG nucleic acid at a proprietary concentration. Samples for testing were prepared by adding pathogen transport buffer containing the desired control material.

[0123] Table 1 below details the CT and NG probes and primers used in the spike assay. [Table 1-1] [Table 1-2]

[0124] In the first set of singleplex assays, samples were spiked with various concentrations of combined NG / CT controls (i.e., Helix Elite™ CT / NG control swabs or CT / NG control nucleic acids) in TE buffer. Combined samples using both Helix Elite™ CT / NG control swabs or CT / NG control nucleic acids were also prepared. RNP was used as a negative control. A "random patient specimen" sample was also prepared using swabs from subjects with neither CT nor NG infections.

[0125] The first set of singleplex assays was performed using primers and probes for NG and RNP, and the second set of singleplex assays was performed using primers and probes for CT and RNP.

[0126] FIG. 6 presents the qPCR readouts from these assays.

[0127] FIG. 7 presents qPCR Cq data and components used in the NG singleplex assay. As shown by their Cq values, samples made using CT / NG nucleic acid controls provided detectable target NG DNA. The lack of corresponding RNP negative control signal indicates that only NG target DNA was being detected. Similarly, samples made using Helix Elite™ CT / NG control swabs provided detectable target NG DNA, indicating that mucosal swab samples can be used to detect STIs using the methods of the present invention. The lack of detectable NG signal upon addition of non-infected patient samples (and corresponding RNP signal) indicates that only target NG DNA was detected. Similar accurate results were obtained when a combined sample was made using both Helix Elite™ CT / NG control swabs and CT / NG control nucleic acids. Furthermore, the Cq values ​​for the combined samples were higher than for either the Helix Elite™ CT / NG control swabs or the CT / NG control nucleic acid samples alone.

[0128] FIG. 8 presents qPCR Cq data and components used in the CT singleplex assay. As shown by their Cq values, samples made using the CT / NG nucleic acid control provided detectable target CT DNA. The lack of corresponding RNP negative control signal indicates that only CT target DNA was being detected. Similarly, samples made using the Helix Elite™ CT / NG control swab provided detectable target CT DNA, indicating that STIs can be detected using mucosal swab samples using the methods of the present invention. The lack of detectable CT signal upon addition of non-infected patient samples (and corresponding RNP signal) indicates that only target CT DNA was detected. Similar accurate results were obtained when combined samples were made using both the Helix Elite™ CT / NG control swab and the CT / NG control nucleic acid.

[0129] After confirming that the assay worked in a singleplex format (i.e., detecting one pathogen from a sample at a time), experiments were performed to evaluate the ability of the methods disclosed herein to detect multiple sexually transmitted pathogens in a single sample using a multiplex format.

[0130] Similar samples to the singleplex assays were prepared using CT / NG controls. Singleplex assays for CT and singleplex assays for NG were performed on these new samples. These new samples were also used in assays using both CT and NG primers and probes in the same sample to evaluate multiplex detection of multiple pathogens using a single sample.

[0131] FIG. 9 presents the qPCR readouts from these assays.

[0132] Figures 10-11 present the qPCR Cq data and components used in the CT and NG singleplex assays. As shown, the data is consistent with the data obtained in the singleplex assays described above.

[0133] Figure 12 presents the qPCR Cq data and components used in the CT and NG multiplex assay. As shown, both CT and NG were easily detected from the same sample by using both CT and NG primers and probes.

[0134] Figure 13 presents a chart summarizing the results obtained in both the singleplex and multiplex assays. As shown, for both NG and CT detection, the results for each sample type were consistent between assays, and the assays were performed on separate days using new samples for each assay.

[0135] Thus, as demonstrated, the methods of the present invention are capable of detecting sexually transmitted pathogens from both swab samples and bodily fluid samples. Additionally, the methods of the present invention are capable of detecting multiple separate sexually transmitted pathogens using a single sample. Additionally, the methods of the present invention.

[0136] (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.

[0137] (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. 1. A method for analyzing nucleic acids without extraction, comprising: contacting a sample with a buffer composition comprising nuclease-free water, an antifungal agent, an antibiotic, and a ribonuclease inhibitor, wherein the sample comprises a mucosal swab sample and / or a bodily fluid sample from a subject; amplifying nucleic acids directly from the sample in the buffer solution with primers specific for one or more target nucleic acids of one or more sexually transmitted pathogens without prior extraction of the nucleic acids; and analyzing the amplicons produced in the amplifying step to detect one or more sexually transmitted pathogens in the subject. A method comprising:

2. 10. The method of claim 1, wherein the mucosal swab sample comprises one or more of a vaginal swab, a cervical swab, a urethral swab, a genital swab, an oral swab, a throat swab, a nasal swab, an eye swab, and any combination thereof.

3. 3. The method of claim 2, wherein the mucosal swab sample comprises two or more of a vaginal swab, a cervical swab, a urethral swab, a genital swab, an oral swab, a pharyngeal swab, a nasal swab, an ocular swab, and combinations thereof.

4. The method of claim 2 , wherein the sample comprises a mucosal sample and a body fluid sample.

5. 5. The method of claim 4, wherein the bodily fluid sample comprises one or more of mucus, blood, plasma, serum, serum derivatives, bile, maternal blood, phlegm, saliva, sputum, sweat, amniotic fluid, menstrual fluid, mammary fluid, follicular fluid, fallopian tube fluid, peritoneal fluid, urine, semen, cerebrospinal fluid (CSF), or a combination thereof.

6. The method of claim 5 , wherein the bodily fluid sample comprises urine.

7. The method of claim 5 , wherein the bodily fluid sample comprises saliva.

8. 10. The method of claim 1, wherein the sample comprises a bodily fluid sample comprising one or more of mucus, blood, plasma, serum, a serum derivative, bile, maternal blood, phlegm, saliva, sputum, sweat, amniotic fluid, menstrual fluid, mammary fluid, follicular fluid, fallopian tube fluid, peritoneal fluid, urine, semen, cerebrospinal fluid (CSF), or a combination thereof.

9. 10. The method of claim 1, wherein the one or more sexually transmitted pathogens are viruses and / or bacteria.

10. 10. The method of claim 9, wherein the one or more sexually transmitted pathogens comprise at least one of bacterial vaginosis, Chlamydia trachomatis (CT), cystitis, Neisseria gonorrhoeae (NG), hepatitis A, hepatitis B, hepatitis C, herpes (herpes simplex type 1 and herpes simplex type 2), HIV, HPV, MPV, lymphogranuloma venereum, molluscum contagiosum, nongonococcal urethritis, pelvic peritonitis, pubic lice, syphilis, trichomoniasis, and vaginitis.

11. 11. The method of claim 10, wherein the target nucleic acids are derived from multiple sexually transmitted pathogens.

12. 12. The method of claim 11, wherein the plurality of sexually transmitted pathogens comprises CT and / or NG.

13. 13. The method of claim 12, wherein the nucleic acid-specific primers comprise one or more primers having a sequence at least 75% identical to the nucleotide sequence set forth in any one of SEQ ID NOs: 1, 2, 4, and 5.

14. Before the mixing process, placing a mucosal swab into said bodily fluid sample in a container; The method of claim 1 , comprising:

15. The method of claim 1 , wherein the nucleic acid is RNA or DNA.

16. 10. The method of claim 1, wherein the analyzing step comprises sequencing the amplicon.

17. The method of claim 1 , wherein the buffer composition comprises a reducing agent.

18. 18. The method of claim 17, wherein the reducing agent is tris(2-carboxyethyl)phosphine hydrochloride solution.

19. 10. The method of claim 1, wherein the antifungal agent comprises amphotericin B and the antibiotic comprises penicillin-streptomycin.

20. The method of claim 1 , wherein the buffer composition stabilizes the nucleic acids from the sample.

21. comparing the amount of target nucleic acid in multiple samples obtained from said subject at sequential time points. wherein an increase or decrease in the amount of the nucleic acid over time indicates disease progression.

22. The method of claim 1 , wherein the amplicon is derived from a tumor virus.

23. The method of claim 1 , wherein the target nucleic acid is an oncogene or a portion thereof.

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