Three-stage nested amplification and multi-stage detection

Abstract

The present disclosure provides methods of three-stage nested amplification that facilitate multiplex amplification and multi-stage detection, in particular multiplex amplification assays that employ target-specific melting probes and / or high resolution melting (HRM) analysis using embedded dyes.

Description

[0001] Cross-references to related applications

[0002] This application claims the benefit of U.S. Provisional Application No. 63 / 539,080, filed September 18, 2023, which is incorporated herein by reference in its entirety. Technical Field

[0003] This disclosure generally relates to methods for detecting target nucleic acids in multiplex nucleic acid amplification-based assays, which can be performed in a kit. The method is suitable for detecting, for example, mutations that confuse drug resistance, and for detecting multiple different organisms in a single assay kit. Background Technology

[0004] Extensively drug-resistant tuberculosis (XDR-TB) is a form of tuberculosis caused by bacteria resistant to some of the most effective anti-TB drugs. XDR-T strains emerge as a result of mismanagement of individuals with multidrug-resistant TB (MDR-TB).

[0005] Nearly one in four people in the world are infected with TB bacteria. People only develop TB when the bacteria become active. This activity can be caused by any factor that weakens a person's immune system, such as HIV, aging, or certain medical conditions. TB is typically treated with a course of four standard or first-line anti-TB drugs: isoniazid, rifampin, ethambutol, and pyrazinamide. Multidrug-resistant TB (MDR-TB) can occur if these drugs are misused or mismanaged. Treatment of MDR-TB with second-line drugs ("SLIDs," namely fluoroquinolones (FLQ) and amikacin, kanamycin, or capreomycin, or newer oral regimens such as bedaquline, pretomanid, and linezolid) requires longer treatment, is more expensive, and has more side effects. XDR-TB occurs when these second-line drugs are also abused or mismanaged and become ineffective. The World Health Organization (WHO) has defined XDR-TB as MDR-TB, which is resistant to at least one fluoroquinolone and SLID. Recently, the oral drugs bedaquiline (BDQ) and linezolid (LZD) have replaced SLIDs as the preferred treatment for drug-resistant TB, and the definition of XDR-TB has changed accordingly (resistance to FLQ and any oral drug).

[0006] TB drug resistance genes have been identified, and resistance mutations to rifampin, isoniazid, and FLQ can be detected in sputum. The mechanisms of resistance to BDQ and LZD are not fully understood. However, based on current evidence, approximately 90% of observed LZD resistance is attributable to a single mutation (C154R) in the rplC gene, with the remaining 2% to 10% attributable to mutations in the rrl (23S rRNA) gene. Resistance to BDQ can occur due to multiple mutations distributed across several genes; mutations in the atpE and Rv0678 genes are the most critical. The atpE gene contains BDQ-R-related mutations in two hotspot regions at codons 28 and 63, and other mutations described at codons 59, 61, and 66 may also be associated with BDQ-R. However, the BDQ-R mutation in the Rv0678 gene is more widespread and can occur anywhere within the 498 bp gene. Summary of the Invention

[0007] The various implementation schemes contemplated herein may include, but are not limited to, one or more of the following:

[0008] Implementation Scheme 1: A method for detecting target nucleic acids in a sample by nucleic acid amplification, the method comprising: contacting sample nucleic acids with a set of pre-amplification primer pairs for amplifying target nucleic acids, wherein the target nucleic acids comprise a first set of target nucleic acids and a second set of target nucleic acids; subjecting the sample nucleic acids and the pre-amplification primer pairs to amplification conditions in solution to amplify any target nucleic acids present in the sample nucleic acids, thereby generating a set of double-stranded amplicones; contacting at least a first portion of the double-stranded amplicones with a first set of nested primers and a first set of target-specific probes in solution to form a first reaction mixture; subjecting the first reaction mixture to asymmetric or symmetric amplification conditions in a first amplification to amplify any of the present first set of target nucleic acids, and performing a first detection, including either helical analysis or real-time analysis, to detect the presence of any of the present first set of target nucleic acids; in solution... At least a second portion of the double-stranded amplicon is contacted with: a second set of nested symmetrical primers for amplifying the second target nucleic acid and a DNA intercalation dye; or, respectively, a second set of nested symmetrical or asymmetric primers for amplifying the second set of target nucleic acids and a second set of target-specific real-time or melting probes; to form a second reaction mixture; in a second amplification, the second reaction mixture is subjected to amplification conditions to amplify the second target nucleic acid or the second set of target nucleic acids in the presence of the second target nucleic acid, and a second detection is performed, the second detection comprising: high-resolution melting analysis to detect the second target nucleic acid in the presence of the second target nucleic acid; or melting analysis or real-time analysis to detect the presence of any of the second set of target nucleic acids present; wherein the first amplification and the first detection, and the second amplification and the second detection are performed sequentially, but may be performed in any order.

[0009] Implementation Scheme 2: A kit for detecting target nucleic acids in a sample, the kit comprising: a body containing a plurality of chambers, wherein the plurality of chambers includes: a sample chamber having at least one fluid outlet in fluid communication with another chamber of the plurality; an optional lysis chamber in fluid communication with the sample chamber, wherein optionally the sample chamber and the lysis chamber are the same; a pre-amplification reagent chamber containing a first set of pre-amplification primer pairs for amplifying the target nucleic acid, wherein the target nucleic acid comprises a first set of target nucleic acids and a second set of target nucleic acids; a first reagent chamber containing a first set of nested primers and a first set of target-specific probes for performing a first amplification; and a second reagent chamber containing a second set of nested primers, wherein... The second set of nested primers contains symmetrical primers for amplifying a second target nucleic acid, and the second reagent chamber further contains a DNA intercalation dye; or the second set of nested primers contains primers for amplifying a second set of target nucleic acids and a second set of target-specific probes; a reaction container fluidly connected to a plurality of chambers of the cassette, and configured for: i) amplification of nucleic acids and ii) detection and identification of one or more amplified products by stranding analysis and / or real-time PCR, wherein the cassette is configured for multi-stage detection; and a filter disposed in a fluid path between the lysis chamber and the reaction container, or in a fluid path between the sample chamber and the reaction container.

[0010] Implementation Scheme 3: The box described in Implementation Scheme 2, wherein the lysis chamber contains one or more lysis reagents for releasing nucleic acids.

[0011] Implementation Scheme 4: The box described in Implementation Scheme 2 or Implementation Scheme 3, wherein the sample chamber and the lysis chamber are the same.

[0012] Implementation Scheme 5: The box described in any one of Implementation Schemes 2 to 4, wherein the box is a box that complies with the Clinical Laboratory Improvement Amendments (CLIA).

[0013] Implementation Scheme 6: A box-based method for detecting target nucleic acids in a sample in a box according to Implementation Scheme 2, the method comprising: placing the sample in a sample chamber of the box; if the sample contains cells, lysing the cells in the sample with one or more lysis reagents present in at least one of the plurality of chambers, or capturing the cells in a filter within the box and lysing the cells by sonication to release sample nucleic acids; if the sample contains cell-free nucleic acids, capturing the free nucleic acids in a nucleic acid capture chamber and eluting the captured nucleic acids after washing to remove impurities; Within the reaction vessel, sample nucleic acids are contacted with pre-amplified primer pairs; the sample nucleic acids and pre-amplified primer pairs are subjected to amplification conditions in solution to amplify any target nucleic acids present in the sample nucleic acids, thereby generating a set of double-stranded amplicones; the double-stranded amplicones are allowed to flow into the first chamber of the plurality of chambers; a first portion of the double-stranded amplicones is extracted from the first chamber of the plurality of chambers and contacted in solution with a first set of nested primers and a first set of target-specific probes to form a first reaction mixture within the reaction vessel; the first reaction mixture is subjected to amplification conditions in a first amplification to amplify... The presence of any of the first set of target nucleic acids is detected by a first detection, including helical analysis or real-time analysis; the first reaction mixture is allowed to flow into at least one of the plurality of chambers, and the reaction vessel is washed; a second portion of the double-stranded amplicon is extracted from the first chamber of the plurality of chambers, and the second portion of the double-stranded amplicon is contacted in solution with: a second set of nested symmetric primers for amplifying the second target nucleic acid and a DNA intercalation dye; or a second set of nested primers for amplifying the second set of target nucleic acids and a second set of target-specific probes; to form a second reaction mixture within the reaction vessel; in a second amplification, the second reaction mixture is subjected to amplification conditions to amplify the second target nucleic acid or the second set of target nucleic acids in the presence of the second target nucleic acid, and a second detection is performed, the second detection including: high-resolution helical analysis to detect the second target nucleic acid in the presence of the second target nucleic acid; or helical analysis or real-time analysis to detect the presence of any of the second set of target nucleic acids; wherein the first amplification and the first detection, and the second amplification and the second detection are performed sequentially, but may be performed in any order.

[0014] Implementation Scheme 7: The method of Implementation Scheme 1, the box of any one of Implementation Schemes 2 to 5, or the box-based method of Implementation Scheme 6, wherein the second set of nested primers comprises symmetric primers for amplifying the second target nucleic acid, accompanied by the DNA intercalation dye, and the high-resolution melting analysis is performed, or the box is configured to perform high-resolution melting analysis to detect the second target nucleic acid in its presence.

[0015] Implementation Scheme 8: The method of Implementation Scheme 1, the box of any one of Implementation Schemes 2 to 5, or the box-based method of Implementation Scheme 6, wherein the second set of nested primers comprises asymmetric primers for amplifying the second set of target nucleic acids, accompanied by the second set of target-specific probes, the second set of target-specific probes being unwinding detection probes, and performing unwinding analysis, or the box is configured to perform unwinding analysis to detect the presence of any of the second set of target nucleic acids present.

[0016] Implementation Scheme 9: The method of Implementation Scheme 1, the box of any one of Implementation Schemes 2 to 5, or the box-based method of Implementation Scheme 6, wherein the second set of nested primers comprises symmetrical primers for amplifying the second set of target nucleic acids, and is accompanied by the second set of target-specific probes, the second set of target-specific probes being real-time probes and being analyzed in real time, or the box is configured to perform real-time analysis to detect the presence of any of the second set of target nucleic acids present.

[0017] Implementation Scheme 10: The method, cassette, or cassette-based method of any one of Implementation Schemes 8 to 9, wherein the method detects 10 to 20 target nucleic acids in each of the first reaction mixture or the second reaction mixture, or the cassette is configured to detect 10 to 20 target nucleic acids in each of the first reaction mixture or the second reaction mixture.

[0018] Implementation Scheme 11: The method, cassette, or cassette-based method of Implementation Scheme 10, wherein the method detects 18 to 20 target nucleic acids in each of the first reaction mixture or the second reaction mixture, or the cassette is configured to detect 18 to 20 target nucleic acids in each of the first reaction mixture or the second reaction mixture.

[0019] Implementation Scheme 12: The method, box, or box-based method of Implementation Scheme 11, wherein the method detects 36 to 40 target nucleic acids in a single box, or the box is configured to detect 36 to 40 target nucleic acids in a single box.

[0020] Implementation Scheme 13: The kit of any one of Implementation Schemes 10 to 12, wherein the reaction container comprises a reaction chamber, and the detection of the target nucleic acid is performed in the reaction chamber.

[0021] Implementation Scheme 14: The kit of any one of Implementation Schemes 10 to 12, wherein the reaction container comprises up to four reaction chambers, and the detection of the target nucleic acid is performed in the up to four reaction chambers.

[0022] Implementation Scheme 15: The method, cassette, or cassette-based method of any one of Implementation Schemes 8 to 10, wherein the pre-amplification primers comprise primers specific to one or more of the following drug resistance genes: amikacin resistance gene rrs; aminoglycoside resistance genes rrs and eis; bedaquiline resistance genes atpE and Rv0678; fluoroquinolone resistance genes gyrA and gyrB; capreomycin resistance genes gidB, rrs, and tlyA; clofazimine resistance gene Rv0 678; delamanid resistance genes fbiA and ddn; ethionamide resistance genes inhA promoter and ethA; ethambutol resistance gene embB; isoniazid resistance genes fabG1, inhA promoter and katG; linezolid resistance genes rplC and rrl; pyrazinamide resistance gene pncA; rifampicin resistance gene rpoB; and streptomycin resistance genes gidB, rrs and rpsL.

[0023] Implementation Scheme 16: The method, cassette, or cassette-based method of any one of Implementation Schemes 8 to 10, wherein the pre-amplification primers comprise primers specific to one or more of the following: Mycobacterium tuberculosis rpoB gene (targeting rpoB RRDR, which contains codons 426 to 452, and mutations in codons 170 and 491), IS6110 gene, IS1081 gene, fabG1 gene, inhA promoter, katG gene, gyrA gene, gyrB gene, pncA gene, rplC gene, rrl gene, atpE gene, and Rv0678 gene.

[0024] Implementation Scheme 17: The method, cassette, or cassette-based method of Implementation Scheme 16, wherein the pre-amplification primers comprise primers specific to each of the following: Mycobacterium tuberculosis rpoB gene (targeting rpoB RRDR, which contains codons 426 to 452, and mutations in codons 170 and 491), IS6110 gene, IS1081 gene, fabG1 gene, inhA promoter, katG gene, gyrA gene, gyrB gene, pncA gene, rplC gene, rrl gene, atpE gene, and Rv0678 gene.

[0025] Implementation Scheme 18: The method, cassette, or cassette-based method of Implementation Scheme 16 or Implementation Scheme 17, wherein the first set of nested primers and the first set of target-specific probes comprise primers and probes specific to one or more of the following: rpoB gene (targeting rpoB RRDR, which contains codons 426 to 452, and mutations in codons 170 and 491), IS6110 gene, IS1081 gene, fabG1 gene, inhA promoter, katG gene, gyrA gene, and gyrB gene.

[0026] Implementation Scheme 19: The method, cassette, or cassette-based method of Implementation Scheme 17, wherein the first set of nested primers and the first set of target-specific probes comprise primers and probes specific to each of the following: rpoB gene (targeting rpoB RRDR, which contains codons 426 to 452, and mutations in codons 170 and 491), IS6110 gene, IS1081 gene, fabG1 gene, inhA promoter, katG gene, gyrA gene, and gyrB gene.

[0027] Implementation Scheme 20: The method, box, or box-based method of any one of Implementation Schemes 16 to 19, wherein the second set of nested symmetrical primers is specific for the rpo0678 gene of Mycobacterium tuberculosis.

[0028] Implementation Scheme 21: The method, box, or box-based method of any one of Implementation Schemes 16 to 19, wherein the embedded dye comprises SYBR green.

[0029] Implementation Scheme 22: The method, cassette, or cassette-based method of Implementation Scheme 16 or Implementation Scheme 17, wherein the first set of nested primers and the first set of target-specific probes comprise primers and probes specific to one or more of the following: rpoB RRDR and rpoB codon 170 target of the rpoB gene of Mycobacterium tuberculosis, IS6110 gene, IS1081 gene, inhA promoter, and katG gene.

[0030] Implementation Scheme 23: The method, cassette, or cassette-based method of Implementation Scheme 22, wherein the first set of nested primers and the first set of target-specific probes comprise primers and probes specific to each of the following: rpoB RRDR and rpoB codon 170 target of the rpoB gene of Mycobacterium tuberculosis, IS6110 gene, IS1081 gene, inhA promoter, and katG gene.

[0031] Implementation Scheme 24: The method, cassette, or cassette-based method of any one of Implementation Schemes 16 to 19, 22, and 23, wherein the second set of nested primers and the second set of target-specific probes comprise primers and probes specific to one or more of the following: the rpoB codon 491 target of the rpoB gene of Mycobacterium tuberculosis, the fabG1 gene, the gyrA gene, and the gyrB gene.

[0032] Implementation Scheme 25: The method, cassette, or cassette-based method of Implementation Scheme 24, wherein the second set of nested primers and the second set of target-specific probes comprise primers and probes specific to each of the following: the rpoB codon 491 target of the rpoB gene of Mycobacterium tuberculosis, the fabG1 gene, the gyrA gene, and the gyrB gene.

[0033] Implementation Scheme 26: The method, box, or box-based method of Implementation Schemes 8 to 25, wherein at least one of the primers and / or probes contains a detectable marker.

[0034] Implementation Scheme 27: The method, box, or box-based method of Implementation Scheme 26, wherein at least one probe, optionally each probe, comprises a fluorescent dye and a quencher molecule.

[0035] Implementation Scheme 28: The method, cassette, or cassette-based method of Implementation Schemes 8 to 27, wherein the method uses and / or the cassette contains primer pairs that selectively hybridize with an exogenous control and / or an endogenous control, wherein the exogenous control is a sample treatment control and wherein the endogenous control is a sample adequacy control.

[0036] Implementation Scheme 29: The method, cassette, or cassette-based method described in Implementation Schemes 8 to 28, wherein the amplification comprises isothermal amplification.

[0037] Implementation Scheme 30: The method, cassette, or cassette-based method of Implementation Schemes 8 to 28, wherein the amplification comprises non-isothermal amplification, optionally through thermal cycling or temperature oscillation.

[0038] Implementation Scheme 31: The method described in Implementation Scheme 1 or the box-based method described in Implementation Scheme 6, wherein the sample is an untreated or digested, purified and concentrated sputum sample, nasal aspirate sample, nasal wash sample, nasal swab sample, nasopharyngeal swab sample, saliva sample, oropharyngeal swab sample, pharyngeal swab sample, bronchoalveolar lavage sample, bronchial aspirate sample, bronchial wash sample, tracheal aspirate sample, tracheal wash sample, tracheal aspirate sample, nasal secretion sample, mucus sample, pleural effusion sample, cerebrospinal fluid sample, fecal sample, tissue biopsy sample, respiratory sample, or a combination thereof.

[0039] Implementation Scheme 32: The method of Implementation Scheme 1 or the box-based method of Implementation Scheme 6, wherein the detection is performed at the same facility from which samples are collected from the object.

[0040] Implementation Scheme 33: The method or box-based method described in Implementation Scheme 32, wherein the method is a point-of-care method.

[0041] Implementation Scheme 34: The method described in Implementation Scheme 1 or the box-based method described in Implementation Scheme 6, wherein the method is performed in a hospital, emergency care center, emergency room, physician's office, health clinic, or home.

[0042] Implementation Scheme 35: The method described in Implementation Scheme 1 or the box-based method described in Implementation Scheme 6, wherein the method is a Clinical Laboratory Improvement Amendment (CLIA) - Exemption Test.

[0043] Implementation Scheme 36: The box-based method described in Implementation Scheme 6, wherein the box is a Clinical Laboratory Improvement Amendment (CLIA) compliant box that operates in accordance with CLIA, is operated by a CLIA-compliant laboratory, or is operated in a CLIA-compliant location.

[0044] Implementation Scheme 37: The method of Implementation Scheme 1, the box of Implementation Scheme 2, or the box-based method of Implementation Scheme 6, wherein the method includes and / or the box facilitates the detection of target nucleic acids in the sample within 150 minutes, 140 minutes, 130 minutes, or 120 minutes of collecting the sample from the subject.

[0045] Implementation Scheme 38: The box described in Implementation Scheme 2 or the box-based method described in Implementation Scheme 6, wherein the box facilitates and / or the method includes detecting the target nucleic acid in the sample within 130 minutes, 120 minutes or 110 minutes from the time the sample is placed in the box.

[0046] Implementation Scheme 39: The box described in Implementation Scheme 3 or the method described in Implementation Scheme 6, wherein the one or more lysis reagents comprise a liquid release agent, a chelating agent, a buffer, and a detergent.

[0047] Implementation Scheme 40: The box or method described in Implementation Scheme 39, wherein the liquid release agent is selected from guanidine thiocyanate, guanidine hydrochloride, alkali metal perchlorate, alkali metal iodide, urea, formamide, or a combination thereof.

[0048] Implementation Scheme 41: The box or method described in Implementation Scheme 39 or 40, wherein the one or more lysis reagents comprise a guanidine salt compound, sodium hydroxide, EDTA, a buffer, and a detergent.

[0049] Implementation Scheme 42: The box described in Implementation Scheme 2, wherein the filter is configured to bind the nucleic acid to be analyzed.

[0050] Implementation Scheme 43: The box of Implementation Scheme 42, wherein the filter comprises glass fiber and optionally a polymer adhesive, or the glass fiber is optionally modified with: DNA binding ligand, optionally alkylamine, cycloalkylamine, alkoxyamine, polyamine moiety, arylamine, intercalating agent, DNA groove binding agent, peptide, amino acid, protein, or combination thereof.

[0051] Implementation Scheme 44: The cartridge described in Implementation Scheme 42 or Implementation Scheme 43, wherein the filter comprises a glass fiber disc with a thickness of 500 micrometers to 2000 micrometers and a pore size of 0.2 micrometers to 1 micrometer.

[0052] Implementation Scheme 45: The box of any one of Implementation Schemes 42 to 44, wherein the filter is configured to bind unwanted substances and allow the nucleic acid to pass through.

[0053] Implementation Scheme 46: The box described in Implementation Scheme 3 or the method described in Implementation Scheme 6, wherein the box further comprises a binding agent, a washing agent, an elution agent, or a combination thereof.

[0054] Implementation Scheme 47: The kit or method described in Implementation Scheme 46, wherein the elution reagent comprises ammonia or an alkali metal hydroxide.

[0055] Implementation Scheme 48: The kit or method described in Implementation Scheme 46 or Implementation Scheme 47, wherein the pH of the elution reagent is higher than about 9, higher than about 10, or higher than about 11.

[0056] Implementation Scheme 49: The kit or method of any one of Implementation Schemes 46 to 48, wherein the elution reagent comprises a polyanionic compound, optionally carrageenan, a carrier nucleic acid or i-carrageenan, and KOH.

[0057] Implementation Scheme 50: The box described in Implementation Scheme 2 or the method described in Implementation Scheme 6, wherein the reaction vessel comprises up to four reaction chambers.

[0058] Implementation Scheme 51: The box described in Implementation Scheme 2 or the method described in Implementation Scheme 6, wherein the reaction vessel comprises a reaction chamber.

[0059] Implementation Scheme 52: The box of Implementation Scheme 2 or the method of Implementation Scheme 6, wherein at least one of the plurality of chambers contains one or more lyophilized reagents.

[0060] Implementation Scheme 53: The box or method described in Implementation Scheme 52, wherein one or more lyophilized reagents are in the form of one or more beads.

[0061] Implementation Scheme 54: The kit or method described in Implementation Scheme 52 or Implementation Scheme 53, wherein one or more lyophilized reagents are selected from primers, probes, salts, dNTPs, thermostable polymerases, reverse transcriptases, or combinations thereof.

[0062] Implementation Scheme 55: The kit or method described in Implementation Scheme 54, wherein one or more lyophilized reagents comprise lyophilized primers and probes.

[0063] Implementation Scheme 56: The box described in Implementation Scheme 2 or the method described in Implementation Scheme 6, wherein the reagents and components in the reaction vessel are in solution. Attached Figure Description

[0064] Figures 1A to 1C show schematic diagrams of sample cartridges with valve assemblies configured to perform various sample processing steps, including chemical lysis of targets, and are configured for PCR and optional integrated nucleic acid analysis. According to some embodiments, Figure 1A shows a sample cartridge housing with a reaction vessel. Figure 1B An exploded view of the sample box is shown, and Figure 1C The components of the valve assembly are shown.

[0065] Figures 2A to 2C Illustrative, non-limiting embodiments of modules and systems (e.g., processing units) for PCR detection and / or quantification of biomarker groups and optional integrated nucleic acid analysis are shown. Figure 2A A module is shown that is configured as a receiving box and interacts with the box's valve assembly to operate the box to facilitate sample preparation and analysis. Figure 2B The module's processing unit (e.g., analytical testing unit) is shown, which interacts with a fluid sample in a reaction vessel to facilitate sample processing and analytical testing of a biomarker set (e.g., PCR and optionally nucleic acid analysis). Figure 2C An analytical system is shown, which has multiple such modules within a housing to receive multiple sample cartridges for testing biomarker groups and / or multiple other targets or groups.

[0066] Figure 3 Various valve assemblies A, B, C, and D are shown, each of which is suitable for one or more types of target lysis, and any of them can be used in the corresponding sample cartridge.

[0067] Figure 4 The layout of the GENEXPERT® box used in the study of Example 1 is shown.

[0068] Figure 5 The experimental setup used in the study of Example 1 is shown, illustrating the biomarkers for drug resistance and the stages in which they were detected.

[0069] Figures 6A to 6D Real-time PCR signals (15 signals) from the study in Example 1 are shown. Figures 6A to 6C The results show that all 15 signals were detected in assays of Mycobacterium bovis BCG (BCG) at 10,000, 1,000, and 100 cfu / mL, respectively. Figure 6D The detection of real-time signals from a sample processing control (SPC) consisting solely of a negative control without BCG is shown.

[0070] Figures 7A to 7D The unchaining signals (13 signals; first derivative) from the study in Example 1 are shown. Figures 7A to 7C The results show that all 13 signals were detected in BCG assays at 10,000, 1,000, and 100 cfu / mL, respectively. Figure 7D The figure shows a melting curve from a negative control that did not contain BCG.

[0071] Figures 8A to 8B The real-time signals of two fluorescent probes and EvaGreen from the studies of amplification 1 and 2 (the second and third stages of the method) in Example 2 are shown respectively. Figure 8A The real-time signal detected in the determination of 10,000 cfu / mL BCG is shown. Figure 8B The results for the negative control are shown, in which only EvaGreen signal was detected, indicating that primer dimers were formed in the absence of any amplification products.

[0072] Figures 9A to 9B The unwinding signal (first derivative) from the study of amplification 1 (the second stage of the method) in Example 2 is shown. Figure 9A The signal was detected in a measurement of 10,000 cfu / mL BCG. Figure 9B The figure shows a melting curve from a negative control that did not contain BCG.

[0073] Figures 10A to 10B The results of high-resolution melt (HRM) from the study of amplification 2 (the third stage of the method) in Example 2 are shown. Figure 10A A suitable melting signal was detected in the determination of 10,000 cfu / mL BCG (N1 to N3 were three replicates). Figure 10B This shows that no suitable signal was detected from the negative control (N1 to N3 were three replicates). Small melting peaks were observed in the negative control. Figure 10B Indicator primer dimer.

[0074] Figures 11A to 11D Results from the study in Example 3 are shown, in which two octa-color / 10-color measurements were performed in the same chamber to generate 18 independent unwinding signals and 2 real-time signals in a 10-color instrument, enabling a total of 20 independent signals to be detected in a 10-color GENEXPERT® instrument. Figure 11A The signal from the nested PCR-1 melting probe is shown; Figure 11B The signal from the nested PCR-1 real-time probe is shown; Figure 11C The signal from the nested PCR-2 denaturation probe was shown; and Figure 11D The signal from the nested PCR-2 real-time probe is shown. Detailed Implementation

[0075] This disclosure describes methods, compositions, apparatus, and systems relying on three-stage nested amplification protocols that selectively and specifically facilitate multiplex target nucleic acid amplification (e.g., polymerase chain reaction [PCR]) and multi-stage detection, which are easily automated and can be used in point-of-care devices. These methods, compositions, apparatus, and systems particularly enable target-specific melting probe detection and high-resolution melting (HRM) analysis in a single automated assay, which can be performed in a single assay kit.

[0076] In single-tube multiplex PCR systems, combining target-specific melting and HRM using intercalating dyes is often impractical, especially when multiple targets are amplified simultaneously. Intercalating dyes are nonspecific and can bind to any amplicon, and therefore have not been previously used to identify variants or mutations in genes within a single cassette. In multiplex PCR systems that amplify several targets simultaneously, HRM with intercalating dyes results in highly complex and uninterpretable melting profiles due to the nonspecific intercalation of the dye into all amplicons amplified in the tube. However, HRM is advantageous for identifying unknown mutations in longer amplicons and mutations distributed across genes >200 bp, as hybridization oligonucleotide melting probes cannot be readily used to cover such large gene fragments for mutation detection. This disclosure overcomes the challenge of combining hybridization target-specific melting detection and intercalating dye HRM detection within the same multiplex PCR system.

[0077] Furthermore, attempts to create multiplex real-time PCR methods have been hampered by the practical problem of simultaneously detecting different nucleic acid sequences in a single sample. A possible approach is to associate different reporter molecules (e.g., fluorescent dyes) with individual amplicons during the PCR reaction, which would allow for the parallel detection of individual reporter molecules by different “colors.” While such methods theoretically offer parallelism, they are limited by: (i) the number of different reporter molecules available; (ii) crosstalk from optical signals present in one channel interfering with optical signals in adjacent channels, and vice versa; and (iii) the availability of imagers and detectors capable of distinguishing different signals.

[0078] Another possible approach to determining a large number of target nucleic acids in a single assay is to separate the target biological sample and physically place it into separate, individual, and isolated amplification chambers using a fluidic system. While this method allows for the simultaneous detection of many targets by performing multiple singleton (i.e., one amplicon per chamber) PCR reactions, it can be suboptimal because it reduces the number of target nucleic acid sequences in each chamber, which can introduce random anomalies (Poisson noise) into the obtained data when the original sample has a low concentration. Furthermore, this method requires complex fluid handling procedures.

[0079] Several benefits of highly multiplexed detection of target nucleic acids in a sample can be achieved by employing analytical platforms such as DNA microarrays or next-generation DNA sequencers. In particular, microarrays are massively parallel, affinity-based biosensors in which target nucleic acids are selectively captured from the same sample at different addressable coordinates (e.g., pixels) on a solid surface. Each addressable coordinate can have a unique capture DNA or RNA probe complementary to the target nucleic acid sequence to be detected in the sample. While microarrays can facilitate the detection of a large number of targets in a sample, they are semi-quantitative and have poor limits of detection (LOD) and detection dynamic range (DDR) due to their endpoint detection nature (i.e., non-real-time detection) and the fact that they lack any target amplification.

[0080] For many applications, automated assays of target nucleic acids are advantageous, which may amplify the challenges discussed above. Assays that can be performed in a single cartridge, which can be used as a point-of-care device and / or in a central laboratory, are of particular interest. However, assays in instruments such as 10-color instruments used for cartridge-based assays can only detect 10 independent optical signals at a time, which presents a practical limitation for multiplex detection.

[0081] The techniques described herein overcome these challenges and have found specific applications where it is advantageous to combine the detection of specific known polymorphisms in the same or different genes with the detection of multiple polymorphisms distributed in a single gene. For example, this detection challenge must be addressed in a single automated assay to identify resistance to the tuberculosis (TB) drugs fluoroquinolones (FLQ), bedaquiline (BDQ), and linezolid (LZD). In one illustrative embodiment, the techniques described herein are capable of detecting more than 10 targets in a single cassette-based assay, with results obtained within 2 hours (e.g., approximately 110 minutes).

[0082] definition

[0083] The term “nucleic acid” refers to a polymer of nucleotides and, unless otherwise limited, includes natural nucleotide analogues that can function in a manner similar to that of naturally occurring nucleotides (e.g., hybridization).

[0084] The term nucleic acid includes any form of DNA or RNA, including, for example, genomic DNA; complementary DNA (cDNA), which is the DNA representation of mRNA, usually obtained by reverse transcription of messenger RNA (mRNA) or by amplification; synthetic or amplified DNA molecules; mRNA; and non-coding RNA.

[0085] The term nucleic acid encompasses both double-stranded and triple-stranded nucleic acid complexes, as well as single-stranded molecules. In double-stranded or triple-stranded nucleic acid complexes, the nucleic acid strands do not need to be mutually extended (i.e., double-stranded nucleic acids do not need to be double-stranded along the entire length of both strands).

[0086] The term "nucleic acid" also encompasses any modifications to it, such as through methylation and / or capping. Nucleic acid modifications can include adding chemical groups to individual nucleic acid bases or to the nucleic acid as a whole, which introduce additional charges, polarizabilities, hydrogen bonds, electrostatic interactions, and functionality. Such modifications can include base modifications, such as sugar modifications at the 2' position, pyrimidine modifications at the 5' position, purine modifications at the 8' position, modifications at the amine of the cytosine ring, substitution of 5-bromouracil, sugar-phosphate backbone modifications, and uncommon base pairing combinations, such as isobasic cytosine and isoguanidine.

[0087] More specifically, in some embodiments, nucleic acids may include polydeoxyribonucleotides (containing 2-deoxy-D-ribose), polynucleotides (containing D-ribose), and any other type of nucleic acid (i.e., N- or C-glycosides of purine or pyrimidine bases), as well as other polymers containing a non-nuclear backbone, such as polyamides (e.g., peptide nucleic acid (PNA)) and polymorpholino polymers (see, for example, Summerton and Weller (1997) “MorpholinoAntisense Oligomers: Design, Preparation, and Properties,” Antisense & Nucleic Acid Drug Dev. 7:1817-195; Okamoto et al. (2002) “Development of electrochemically gene-analyzing method using DNA-modified electrodes,” Nucleic Acids Res. Supplement No.). 2:171-1722), and other synthetic sequence-specific nucleic acid polymers, provided that the polymer contains nucleobases whose configuration allows for base pairing and base stacking, as seen in DNA and RNA, for example. The term nucleic acid also includes locked nucleic acids (LNAs), described in U.S. Patent Nos. 6,794,499, 6,670,461, 6,262,490, and 6,770,748, the disclosures of which are incorporated herein by reference in their entirety.

[0088] Nucleic acids can be derived from completely chemically synthesized processes, such as solid-phase mediated chemical synthesis, from biological sources, such as by isolation from any species that produces nucleic acids, or from processes involving manipulation of nucleic acids using molecular biology tools, such as DNA replication, PCR amplification, reverse transcription, or combinations of those processes.

[0089] In the context of two or more amino acid or nucleotide sequences, the term “sequence identity” means that two or more sequences are identical or have a specified percentage of identical amino acid residues or nucleotides when compared and aligned to achieve maximum correspondence, such as when measured using sequence comparison algorithms or by visual inspection.

[0090] To perform sequence comparisons to determine the percentage of nucleotide or amino acid sequence identity, a sequence is typically used as a "reference sequence" and compared to a "test" sequence. When using a sequence comparison algorithm, the test and reference sequences are input into the computer, subsequence coordinates are specified (if needed), and sequence algorithm program parameters are specified. The sequence comparison algorithm then calculates the percentage sequence identity of the test sequence relative to the reference sequence based on the specified program parameters. Sequence alignment can be performed using BLAST with default parameters.

[0091] The term “gene” as used in this article encompasses coding sequences, introns, and any related control sequences involved in the expression of coding sequences.

[0092] The term "complementarity" as used in this article refers to the ability of two nucleotides to pair precisely; that is, if a nucleotide at a given position in one nucleic acid can form a hydrogen bond with a nucleotide in another nucleic acid to form a canonical base pair, then the two nucleic acids are considered complementary at that position. Complementarity between two single-stranded nucleic acid molecules can be "partial," where only some nucleotides bind, or it can be complete when there is perfect complementarity between the single-stranded molecules. The degree of complementarity between nucleic acid strands has a significant impact on the efficiency and strength of hybridization between nucleic acid strands.

[0093] "Selective hybridization" or "selective annealing" refers to the binding of a nucleic acid to a target nucleic acid under strict, defined conditions, while there is no substantial binding to other nucleic acids present in the hybridization mixture. Those skilled in the art will recognize that relaxing the stringency of hybridization conditions allows for tolerance of sequence mismatches.

[0094] In some implementations, hybridization is performed under stringent hybridization conditions. The phrase "stringent hybridization conditions" typically refers to conditions with defined ionic strength and pH, compared to the melting temperature (T0) for a specific sequence. m Temperatures approximately 5°C lower than to approximately 20°C or 25°C. The T used in this article... m It is the temperature at which a group of double-stranded nucleic acid molecules partially dissociates into single strands. It is used to calculate the T0 of nucleic acids. mThe method is well known in the art (see, for example, Berger and Kimmel (1987) Methods in Enzymology, Vol. 152: Guide to Molecular Cloning Techniques, San Diego: Academic Press, Inc. and Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual, 2nd ed., Vols. 1–3, Cold Spring Harbor Laboratory), both of which describe the stringent hybridization conditions and are incorporated herein by reference). As indicated by the standard references, T m A simple estimate of the value can be calculated using the following formula: When nucleic acids are in an aqueous solution of 1 M NaCl, T m =81.5 + 0.41 (% G+C) (see, for example, Anderson and Young, Quantitative Filter Hybridization in Nucleic Acid Hybridization (1985)). The melting temperature of the hybrid (and therefore the conditions for rigorous hybridization) is influenced by a variety of factors, such as the length and properties of the primers or probes (DNA, RNA, base composition) and the properties of the target nucleic acid (DNA, RNA, base composition, presence in solution or immobilization, etc.), as well as the concentrations of salts and other components (e.g., the presence or absence of formamide, dextran sulfate, polyethylene glycol). The effects of these factors are well known and discussed in standard references in the art. Exemplary rigorous conditions suitable for achieving most sequence-specific hybridizations are a temperature of at least about 60°C and a salt concentration of about 0.2 mol at pH 7. T based on nearest-neighbor thermodynamics of oligonucleotide sequences m The calculations can be performed as described in “A unified view of polymer, dumbbell, and oligonucleotide DNA nearest-neighbor thermodynamics” John SantaLucia, Jr., PNAS February 17, 1998, Vol. 95, No. 4, pp. 1460-1465 (for which this description is incorporated herein by reference).

[0095] The term "oligonucleotide" is used to refer to relatively short nucleic acids, typically shorter than 200 nucleotides, more specifically, shorter than 100 nucleotides, and most specifically, shorter than 50 nucleotides. Generally speaking, oligonucleotides are single-stranded DNA molecules.

[0096] The term "primer" refers to an oligonucleotide capable of hybridizing (also known as "annealing") with nucleic acids and acting as an initiation site for nucleotide (RNA or DNA) polymerization under appropriate conditions (i.e., the presence of four different nucleosides and reagents for polymerization, such as DNA or RNA polymerases or reverse transcriptases), in an appropriate buffer, and at an appropriate temperature. The appropriate length of a primer depends on its intended use, but primers are typically at least 7 nucleotides long, and in some embodiments, are 10 to 30 nucleotides long, or in some embodiments, 10 to 60 nucleotides long. In some embodiments, primers may be, for example, 15 to 50 nucleotides long. Short primer molecules generally require lower temperatures to form a sufficiently stable hybridization complex with the template. Primers do not need to reflect the exact sequence of the template, but must be sufficiently complementary to hybridize with it.

[0097] If a primer or a portion thereof hybridizes with a nucleotide sequence within another nucleic acid, the primer is said to be "annealed" or "hybridized" with that nucleic acid. Statements stating that a primer hybridizes with a specific nucleotide sequence are not intended to imply that the primer hybridizes completely or exclusively with that nucleotide sequence. For example, in some embodiments, amplification primers used herein are referred to as "annealed to a nucleotide sequence" or "specific to a nucleotide sequence." This description covers primers that are completely annealed to a nucleotide sequence, as well as primers that are partially annealed to a nucleotide sequence.

[0098] The term "primer pair" refers to a set of primers, including a 5' "upstream primer" or "forward primer" that hybridizes to the 5' complementary sequence of the DNA sequence to be amplified, and a 3' "downstream primer" or "reverse primer" that hybridizes to the 3' end of the sequence to be amplified. As those skilled in the art will recognize, the terms "upstream" and "downstream" or "forward" and "reverse" are not intended to be limiting, but rather to provide illustrative directions in some embodiments.

[0099] As used in this article, the term "pre-amplification primer pair" refers to a pair of primers used in the initial amplification followed by at least one subsequent amplification designed to amplify at least one of the same target nucleic acids as in the initial amplification.

[0100] The “nested primers” used in this paper selectively hybridize within amplicon generated by previous amplification (e.g., initial or “pre-amplification”).

[0101] The “symmetric primers” used in this paper generate double-stranded amplicon.

[0102] The “asymmetric primers” used in this paper generate nucleic acid strands complementary to the annealed nucleic acid strands, i.e., asymmetric primers generate single-stranded amplicon.

[0103] A probe is a nucleic acid that can bind to a target nucleic acid with a complementary sequence via one or more types of chemical bonds, typically through complementary base pairing, usually through hydrogen bonding, to form a double-stranded structure. Probes can be labeled with a detectable portion to allow for easy detection, especially once the probe has hybridized to its complementary target. Alternatively, probes can be unlabeled but can be detected by specific binding to directly or indirectly labeled ligands. Probe size can vary significantly.

[0104] The term "specific to nucleic acid" used in this article to refer to a portion of a primer or a nucleotide sequence within a primer refers to a primer or nucleotide sequence that can specifically anneal to the target nucleic acid under suitable annealing conditions.

[0105] The term “target” is used herein with reference to both “target nucleic acid” and “target organism.” The former refers to the nucleic acid to be detected, and the latter refers to the organism to be detected. The term “target nucleic acid” is generally used herein to refer to the nucleic acid fragment defined by primer pairs and producing an amplicon in an amplification reaction; the term “amplification target” is also used herein to refer to this type of target nucleic acid. Primers and probes are also referred to as “target” nucleic acid sequences, and therefore these sequences can also be understood as “target nucleic acids.” Additionally, primers and probes are referred to as “targeting” genes or “gene-specific.” In this usage, primers and probes can be used to detect the presence of a specific gene by specifically hybridizing with a portion of a gene indicating its presence. Those skilled in the art will understand the meaning of “target” and “target nucleic acid” from the context of the use of the term. In some embodiments, multiple target nucleic acids can be detected to detect a single target organism. In some embodiments, a single target nucleic acid can be detected to detect a single target organism. In some embodiments, assays can use multiple target nucleic acids targeting one or more target organisms and a single target nucleic acid targeting one or more different target organisms.

[0106] Amplification according to this teaching encompasses any method that typically replicates at least a portion of at least one target nucleic acid in a template-dependent manner, including but not limited to a wide range of techniques for linear or exponential amplification of nucleic acid sequences. Illustrative methods for performing amplification steps include PCR, nucleic acid strand-based amplification (NASBA), two-step multiplex amplification, rolling circle amplification (RCA), etc., including multiple versions and combinations thereof, such as, but not limited to, OLA / PCR, PCR / OLA, LDR / PCR, PCR / PCR / LDR, PCR / LDR, LCR / PCR, PCR / LCR (also known as combined chain reaction-CCR), helicase-dependent amplification (I), etc. Descriptions of such techniques can be found in the following and other sources: Ausubel et al.; PCR Primer: A Laboratory Manual, Diffenbach, ed., Cold Spring Harbor Press (1995); The Electronic Protocol Book, Chang Bioscience (2002); Msuih et al., J. Clin. Micro. 34:501-07 (1996); The Nucleic Acid Protocols Handbook, R. Rapley, ed., Humana Press, Totowa, NJ (2002); Abramson et al., Curr Opin Biotechnol. Feb. 1993;4(1):41-7, U.S. Patent No. 6,027,998; U.S. Patent No. 6,605,451, Barany et al., PCT Publication No. WO 97 / 31256; Wenz et al., PCT Publication No.WO 01 / 92579; Day et al., Genomics, 29(1): 152-162 (1995); Ehrlich et al., Science252:1643-50 (1991); Innis et al., PCR Protocols: A Guide to Methods and Applications, Academic Press (1990); Favis et al., Nature Biotechnology 18:561-64 (2000); and Rabenau et al., Infection 28:97-102 (2000); Belgrader, Barany, and Lubin, Development of a Multiplex Ligation Detection Reaction DNA Typing Assay, Sixth International Symposium on Human Identification, 1995 (available at a21romegaega.com / geneticidproc / ussymp6proc / blegrad.html); LCR KitInstruction Manual, catalog number 200520, Rev. #050002, Stratagene, 2002; Barany, Proc. Natl. Acad. Sci. USA 88:188-93 (1991); Bi and Sambrook, Nucl. Acids Res. 25:2924-2951 (1997); Zirvi et al., Nucl. Acid Res. 27:e40i-viii (1999); Dean et al., ProcNatl Acad Sci USA 99:5261-66 (2002); Barany and Gelfand, Gene 109:1-11 (1991); Walker et al., Nucl. Acid Res. 20:1691-96 (1992); Polstra et al., BMC Inf. Dis. 2:18-(2002); Lage et al., Genome Res. February 2003; 13(2):294-307, and Landegren et al., Science 241:1077-80 (1988), Demidov, V., Expert Rev Mol Diagn. November 2002; 2(6):542-8.Cook et al., J Microbiol Methods. May 2003; 53(2):165-74; Schweitzer et al., CurrOpin Biotechnol. February 2001; 12(1):21-7; U.S. Patent No. 5,830,711, U.S. Patent No. 6,027,889, U.S. Patent No. 5,686,243, PCT Publication No. WO0056927A3, and PCT Publication No. WO9803673A1.

[0107] In some implementations, amplification comprises at least one cycle of the following sequential procedure: annealing at least one primer to a complementary or substantially complementary sequence in at least one target nucleic acid; synthesizing at least one nucleotide chain in a template-dependent manner using a polymerase; and denaturing the newly formed nucleic acid duplex to separate the strands. This cycle may or may not be repeated. Amplification may involve thermal cycling or may be performed isothermally.

[0108] The term "amplification conditions" as used in this article refers to the conditions that promote the amplification of target nucleic acids in the presence of suitable primers.

[0109] As used in this article, “in solution” means not fixed to any kind of substrate, such as a bead or a surface in a box, like a chamber wall.

[0110] "Multiplex amplification reaction" is a reaction that simultaneously amplifies two or more nucleic acids that can be distinguished by their sequences.

[0111] As used in this article, the term "qPCR" refers to quantitative real-time polymerase chain reaction (PCR), also known as "real-time PCR" or "kinetic polymerase chain reaction"; all terms refer to PCR with real-time signal detection.

[0112] The term "melting curve analysis" refers to the use of the dissociation characteristics of double-stranded nucleic acid fragments during heating. Initially, ultraviolet absorption measurements were used to observe strand dissociation, but fluorescence-based techniques are now the most common method. Temperature-dependent dissociation between two DNA strands can be measured in "melting assays," for example, using DNA-intercalated fluorophores (such as SYBR green or EvaGreen) or fluorophore-labeled DNA probes. In the case of SYBR green (whose fluorescence intensity increases 1000-fold when intercalated into the minor grooves of two DNA strands), DNA dissociation during heating can be measured by the resulting significant decrease in fluorescence. Alternatively, juxtaposed probes (one with a fluorophore and the other with a suitable quencher) can be used to determine the complementarity of the probe with the target nucleic acid sequence.

[0113] "Reagent" broadly refers to any reagent used in a reaction other than the analyte (e.g., the nucleic acid being analyzed). Exemplary reagents used in nucleic acid amplification reactions include, but are not limited to, buffers, metal ions, polymerases, reverse transcriptases, primers, template nucleic acids, nucleotides, labels, dyes, nucleases, dNTPs, etc. Reagents used in enzymatic reactions include, for example, substrates, cofactors, buffers, metal ions, inhibitors, and activators.

[0114] As used herein, the term "label" refers to any atom or molecule that can be used to provide a detectable and / or quantifiable signal. In particular, labels can be directly or indirectly linked to nucleic acids or proteins. Suitable labels that can be linked to probes include, but are not limited to, radioisotopes, fluorophores, chromophores, mass labels, electron-dense particles, magnetic particles, spin labels, chemiluminescent molecules, electrochemically active molecules, enzymes, cofactors, and enzyme substrates.

[0115] The term "dye" as used in this article generally refers to any organic or inorganic molecule that absorbs electromagnetic radiation and produces a detectable signal (such as a fluorescence signal).

[0116] The term "quencher" as used in this article generally refers to any organic or inorganic molecule that reduces the level of a detectable signal.

[0117] The term “detection” as used in this article means “determining the presence of an item,” such as determining the presence of a nucleic acid sequence.

[0118] As used in this paper, the term "multi-stage detection" refers to at least two detection steps performed sequentially. Each detection step may be performed in a single reaction chamber or multiple reaction chambers.

[0119] As used herein, the term "treatment regimen" refers to any medical intervention designed to alleviate the symptoms and / or pathological condition of a disease. A treatment regimen may include one or more actions (e.g., bed rest, increased fluid intake), over-the-counter or prescription medications, supplements, food, beverages, or the use of medical devices (e.g., a ventilator).

[0120] As used herein, “Clinical Laboratory Improvement Amendments (CLIA)” refers to the Clinical Laboratory Improvement Amendments (CLIA) regulations of 1988, effective as of the date of the original filing of this application. CLIA regulations comprise federal standards applicable to all U.S. facilities or sites that test human samples for health assessment or diagnosis, prevention, or treatment of disease. “CLIA-compliant” testing is testing that complies with these regulations. “CLIA-exempt” testing includes testing that does not comply with all of these regulations. For example, CLIA-exempt testing includes testing systems approved by the U.S. Food and Drug Administration for home use, as well as those tests approved for exemption under CLIA standards.

[0121] As used herein, "endogenous control" refers to a portion naturally present in the sample to be used for detection. In some embodiments, the endogenous control is a "sample adequacy control" (SAC), which can be used to determine whether sufficient sample was used in the assay, or whether the sample contains sufficient biological material, such as cells. In some embodiments, the endogenous control is RNA (e.g., mRNA, tRNA, ribosomal RNA, etc.), such as human RNA for human samples. Non-limiting exemplary endogenous controls include ABL mRNA, GUSB mRNA, GAPDH mRNA, TUBB mRNA, and UPKla mRNA. In some embodiments, an endogenous control, such as a SAC, that can be detected in the same manner as the target nucleic acid (e.g., RNA) is selected, and in some embodiments, it is detected simultaneously with the target nucleic acid (e.g., RNA).

[0122] As used herein, "exogenous control" refers to a portion added to a sample or assay, such as a "sample processing control" (SPC). In some embodiments, the assay reagent includes an exogenous control. Typically, an exogenous control is selected that is not expected to be present in the sample to be used for detection, or that is present in the sample at a very low level, such that the amount of the naturally occurring portion in the sample is undetectable, or is detectable at a level much lower than the amount added to the sample as an exogenous control. In some embodiments, the exogenous control comprises a nucleotide sequence that is not expected to be present in the sample type used to detect the target nucleic acid (e.g., RNA). In some embodiments, the exogenous control comprises a nucleotide sequence known to be absent in the species from which the sample was obtained. In some embodiments, the exogenous control comprises a nucleotide sequence from a species different from the object from which the sample was obtained. In some embodiments, the exogenous control comprises a nucleotide sequence known to be absent in any species. In some embodiments, an exogenous control is selected that can be detected in the same manner as the target nucleic acid (e.g., RNA), and in some embodiments, is detected simultaneously with the target nucleic acid (e.g., RNA). In some embodiments, the exogenous control is RNA. In some such implementations, the exogenous control is armed RNA®, which contains RNA packaged in a phage protective layer. See, for example, WalkerPeach et al., Clin. Chem. 45: 12: 2079-2085 (1999).

[0123] Three-stage nested amplification and multi-stage detection

[0124] This disclosure provides a three-stage nested amplification method that facilitates multiplex amplification and multi-stage detection, particularly multiplex amplification assays using target-specific melting probes and / or high-resolution melting (HRM) analysis utilizing intercalating dyes. Two illustrative methods, particularly applicable to automated single-cassette assays, such as those performed by Cepheid's GeneExpert® system, are described below. These methods share the following common steps.

[0125] First, the sample nucleic acid is pre-amplified using pre-amplification primers that specifically amplify the target nucleic acid. The target nucleic acid comprises two “groups” (i.e., “first” group and “second” group) of target nucleic acids, which are detected separately from each other using multi-stage detection steps that can be performed sequentially (in any order). A target nucleic acid “group” may contain multiple target nucleic acids, and one within a group is typically so. In some embodiments, a “group” may consist of only one target nucleic acid. Pre-amplification is typically performed with all reaction components in solution to produce a set of double-stranded amplicones.

[0126] A portion of the double-stranded amplicon (referred to as the “first portion” for ease of discussion) is combined with a first set of nested primers and a first set of target-specific probes to form a first reaction mixture. The nested primers anneal to the sequences present in the double-stranded amplicon corresponding to the first set of target nucleic acids and amplify (typically in solution) one or both strands of the first portion of the double-stranded amplicon (asymmetric primers preferentially produce one strand, while symmetric primers produce two strands, as in standard polymerase chain reaction (PCR)). These copies can be readily detected by using a melting probe assay or a real-time PCR assay using probes present in the first target detection reaction mixture. This amplification enables convenient multiplex detection of the target nucleic acid using melting assays and / or real-time assays, constituting the first detection step in this method using multi-stage detection. The term “first” as used in this context is solely for distinguishing this step from the “second” detection steps described below. These terms are intended to imply that these detections are performed separately and sequentially as part of a multi-stage detection. These terms are not intended to imply any particular order: in this method, the first detection can be performed first or second, and the second detection can be performed second or first. Although the method steps are discussed in relation to the detection of multiple target nucleic acids, those skilled in the art will understand that the method steps can also be used to detect a single target nucleic acid.

[0127] The second portion of the double-stranded amplicon generated during pre-amplification is amplified in the second target detection reaction, subsequently detecting one or more of the second set of target nucleic acids. The use of the terms "first" and "second" does not imply a specific order of these steps: amplification of the first reaction mixture may occur before, after, or simultaneously with amplification of the second reaction mixture (again, the order in which the first and second reaction mixtures are formed is not important). The details of amplification of the second reaction mixture differ between the two illustrative embodiments of the method described herein.

[0128] Illustrative Method A: Pre-amplification Amplification 1 Test 1 Single amplification 2 HRM detection 2

[0129] In one embodiment, the second reaction mixture comprises a second set of nested symmetrical primers for amplifying individual target nucleic acids constituting a second target nucleic acid “set,” and a DNA intercalation dye. High-resolution melting (HRM) analysis is performed to detect and optionally characterize the second target nucleic acid. This detection represents one possible second detection step in an illustrative embodiment of the method.

[0130] In some implementations, amplification 1 is performed using asymmetric primers to generate single-stranded amplicones, and the target-specific probe is a melting probe. In this case, detection 1 can be performed by melting analysis. When all amplification and detection steps in the method are performed in a single reaction chamber, the use of asymmetric primers for amplification 1 offers the advantage of minimal interference from carryover of the asymmetric single-stranded amplicon from amplification 1 (less carryover single-stranded amplicon will be less likely to bind to DNA intercalation dyes compared to double-stranded amplicon generated using symmetric primers in amplification 2). Reduced carryover can provide clearer HRM results in detection 2, as well as higher sensitivity, especially when the second target nucleic acid is large, such as the Rv0678 gene, which is >500 base pairs.

[0131] Illustrative Method B: Pre-amplification Amplification 1 Test 1 Amplification 2 Detection 2

[0132] In another embodiment, the second reaction mixture includes a second set of nested primers for amplifying a second set of target nucleic acids and a second set of target-specific probes. Helicoptering or real-time analysis is performed to detect the presence of any second set of target nucleic acids present. This detection represents another possible second detection step in another illustrative embodiment of the method.

[0133] In some implementations, this method can be performed to detect a large number of target nucleic acids in a single assay. For example, pre-amplification may consist of 20 different pre-amplification primer pairs, which can generate 20 individual amplicons according to a parallel amplification protocol for multiple genes in a single organism, or generate one or more amplicons from a list of 20 target genes present in 20 different organisms; or, a combination of these methods can be used to generate one or more amplicons in 2 to 19 different organisms. A first portion of the amplicons thus generated can be further amplified using 10 nested primer pairs to detect 10 target nucleic acids in Amplification 1 / Detection 1, and a second portion of the amplicons generated by pre-amplification can be further amplified using another 10 nested primer pairs to detect 10 additional target nucleic acids in Amplification 2 / Detection 2. Both detection stages are performed in a single reaction chamber, with the second detection step performed after the first set of 10 amplicons has been removed from and washed away from the reaction chamber.

[0134] Higher levels of multiplexing are possible using a combination of unwinding and real-time probes. For example, the following shows the number of signals that can be detected using Method B in automated single-box assays, such as those performed by Cepheid's GeneExpert® system, using only Taqman probes, only unwinding probes, or a combination of both.

[0135] Table 1: Desolvation and... in cassette-based nucleic acid amplification using 10 optical channels in the GENEXPERT® system Multiple analysis of real-time probes

[0136]

[0137] Assuming each optical channel can detect at least three independent unwinding signals, as has been demonstrated in some existing measurements.

[0138] The total number of detectable signals varies based on the number of available optical channels. For example, in a system with five optical channels, the total number of detectable signals is halved, or in a system with twenty optical channels, it is doubled.

[0139] Given the various problems with currently available multiplex PCR methods, this level of multiplexing represents a significant advancement, especially in automated systems. For example, while multiplexing amplification reactions of large numbers of targets (e.g., multiplex PCR) is possible, simultaneous detection of multiple amplicones is not straightforward. To date, real-time multiplex methods (defined as the process of simultaneously amplifying and detecting multiple nucleic acid sequences in a single reaction chamber) have been implemented for small numbers of amplicones. The efficient multiplexing of assays in the same reaction volume, allowing for multiple parallel target amplification and detection in the same reaction chamber, has generated considerable interest. Such methods not only offer better utilization of raw DNA samples but also significantly reduce any of the complexity associated with fluid dynamics and liquid handling procedures used to run multiple singleton reactions.

[0140] In some embodiments, the methods described herein may include multiplex amplification reactions that can be designed to detect 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 or more target nucleic acids in each amplification reaction mixture. This level of multiplex analysis can be achieved by using primers and probes that do not substantially undergo cross-reaction or off-target binding, and in some embodiments, by combining the unwinding of the same amplification reaction with real-time detection. The methods described herein further extend the number of targets that can be detected in a single assay by using a pre-amplification reaction, two subsequent separate amplification reactions, and multi-stage (in this case, two-stage) detection. The two illustrative embodiments described above can reduce signal interference and artifacts due to remnants and are easily automated, for example, in a cassette-based manner.

[0141] In some implementations, to increase the number of target nucleic acids detected per channel, the following methods can be used: (i) T can be used m (ii) De-annealing probes below the annealing temperature are combined with Taqman and de-annealing probes in the same channel (no amplification curve); and (ii) several de-annealing target nucleic acids in one channel; the de-annealing window for each target will depend on the sequence variation of that target.

[0142] Since a large number of oligonucleotides combined in a reaction mixture can lead to undesirable interactions between them, in some embodiments, modified nucleotides can be used to reduce primer-primer interactions.

[0143] Multiplex detection of tuberculosis drug resistance

[0144] In one implementation, method A described above can be used to detect resistance to the TB drugs FLQ, LZD, and BDQ by targeting, for example, 6 to 7 different genes that can be co-amplified in a single cassette. The presence or absence of mutations in these genes can be detected by monitoring the Tm values ​​of unwinding probes from all target genes except for one selected target gene and performing HRM using a DNA intercalation dye to assess mutations in additional target genes. This probe unwinding and SYBR green HRM multiplex PCR assay can be performed in a single cassette using a three-stage nested PCR, characterized by the following workflow: pre-amplification PCR1 Unchain Uniplex PCR2 HRM. In one illustrative embodiment, pre-amplification may include external primers for all target genes gyrA, gyrB, rplC, rrl, atpE, Rv0678, and IS1081 (optional; for MTB detection) to pre-amplify all targets. PCR1 amplification may include nested asymmetric primers for all targets except Rv0678 and target-specific melting / detection probes. This amplification produces single-stranded DNA (ssDNA) amplicones for all targets except Rv0678. These single-stranded amplicones can be detected in the first stage of detection by melting analysis of the melting probes. PCR2 amplification (which may be a haploid amplification of the Rv0678 gene) may include symmetrical nested primers that produce double-stranded DNA (dsDNA) amplicones for Rv0678. Mutations in these double-stranded DNA amplicones can be detected by HRM in the second stage of detection, enabling the detection of mutations regardless of where they occur in the gene. Using asymmetric primers in PCR1 followed by symmetric primers in PCR facilitated HRM analysis in at least two ways: i) it reduced interference from asymmetric ssDNA amplicon remnants from PCR1 (small amounts of remnant ssDNA amplicons will be less likely to bind to SYBR green), while resulting in clearer Rv0678 assay-specific HRM results due to the preferential generation of double-stranded amplicons in singlet form in PCR2; and ii) it provided better sensitivity for Rv0678 amplicons >500 bp.

[0145] Illustrative primers for multiplex detection of tuberculosis drug resistance. The forward and reverse primers are named "FOR" and "REV," respectively.

[0146] Table 2: Illustrative primers for multiplex detection of tuberculosis drug resistance

[0147]

[0148] Considerations for primers and probes used in practicing the methods described herein are described in more detail in the following section entitled “Exemplary Polynucleotides”.

[0149] Comparison

[0150] In some embodiments, the assay described herein may include the detection of at least one endogenous control in addition to the selected target nucleic acid. In some embodiments, the endogenous control is a Sample Adequacy Control (SAC). The SAC ensures that the sample contains human cells or human DNA. The assay includes primers and probes for detecting a single copy of a human gene. The SAC signal is considered only if the sample is negative for all other targets. A negative SAC indicates the absence of human cells in the sample due to inadequate sample mixing or insufficient sample collection. In some such embodiments, if neither the target nucleic acid nor the SAC is detected in the sample, the assay result is considered "invalid" because the sample may be insufficient. While not intended to be bound by any particular theory, an insufficient sample may be too dilute, contain too little cellular material, or contain assay inhibitors, etc. In some embodiments, failure to detect the SAC may indicate a failed assay reaction. In some embodiments, the endogenous control is RNA (e.g., mRNA, tRNA, ribosomal RNA, etc.). Non-limiting exemplary endogenous controls include ABL mRNA, GUSB mRNA, GAPDH mRNA, TUBB mRNA, and UPKla mRNA.

[0151] In some embodiments, the assay described herein may include the detection of at least one exogenous control in addition to the selected target nucleic acid. In some embodiments, the exogenous control is a sample processing control (SPC). The SPC verifies that the sample processing is adequate. Additionally, this control detects sample-related inhibition in the real-time PCR assay, ensuring that the PCR reaction conditions (temperature and time) are suitable for the amplification reaction and that the PCR reagents are effective. The SPC should be positive in negative samples and can be negative or positive in positive samples. If the SPC meets validated acceptance criteria, the SPC passes. In some such embodiments, if neither the target nucleic acid nor the SPC is detected in the sample, the assay result is considered "invalid" because errors may have occurred in the sample processing, including but not limited to assay failure. Non-limiting exemplary errors in sample processing include inadequate sample processing, the presence of assay inhibitors, the presence of nucleases (e.g., RNases), or damaged reagents. In some embodiments, an exogenous control (e.g., the SPC) is added to the sample. In some embodiments, an exogenous control (e.g., the SPC) is added during the assay, for example, using one or more buffers or reagents. In some implementations, when using the GENEXPERT® system, the SPC may be included in the GENEXPERT® box. In some implementations, the exogenous control (e.g., the SPC) is armed RNA® protected by a phage coat.

[0152] In some embodiments, endogenous and / or exogenous controls are detected simultaneously with the selected target nucleic acid, for example, in the same assay. In some embodiments, the assay includes reagents for simultaneously detecting the target nucleic acid and SAC and / or exogenous controls in the same assay reaction mixture. In some such embodiments, for example, the assay reaction mixture includes primer sets for amplifying the target nucleic acid, primer sets for amplifying the SAC, and / or primer sets for amplifying the exogenous control, and optional labeled probes (e.g., helical probes or TaqMan® probes) for detecting the amplification products.

[0153] In some implementations, the assay includes a probe check control (PCC). In some such implementations, prior to the start of the PCR reaction, the system (e.g., a GENEXPERT system) measures the fluorescence signal from the probe to monitor bead rehydration, reaction tube filling, probe integrity, and dye stability. If the PCC meets validated acceptance criteria, the PCC passes.

[0154] Polynucleotides

[0155] In some embodiments, a polynucleotide is provided for detecting the aforementioned biomarkers. In some embodiments, a synthetic polynucleotide is provided. As used herein, a synthetic polynucleotide refers to a polynucleotide that has been chemically or enzymatically synthesized in vitro. Chemical synthesis of polynucleotides includes, but is not limited to, synthesis using a polynucleotide synthesizer such as OligoPilot. TM (GE Healthcare), ABI 3900 DNA synthesizer (Applied Biosystems), etc. Enzymatic synthesis includes, but is not limited to, the production of polynucleotides through enzymatic amplification (e.g., PCR). Polynucleotides may contain one or more analogs of canonical nucleotides (e.g., modified nucleotides).

[0156] In some implementations, a polynucleotide is provided comprising a region containing at least 6, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, or at least [number missing] of a selected target nucleic acid and / or the exemplary controls discussed above. The 28, at least 29, or at least 30 consecutive nucleotides have at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity, or at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% complementarity.

[0157] In several embodiments, the exemplary polynucleotide comprises at least: 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides. In several embodiments, the polynucleotide comprises fewer than: 200, 150, 100, 50, 40, 30, or 20 nucleotides. In several embodiments, the exemplary polynucleotide has a length of 6 to 200, 8 to 200, 8 to 150, 8 to 100, 8 to 75, 8 to 50, 8 to 40, 8 to 30, 15 to 100, 15 to 75, 15 to 50, 15 to 40, or 15 to 30 nucleotides.

[0158] In some embodiments, detection of each target nucleic acid can be performed using a single labeled primer or probe specific to each target nucleic acid. Different primers and / or probes may have the same label. By using primers or probes labeled with different detectable moieties (e.g., different fluorescent reporter dyes), many target nucleic acids can be detected simultaneously in a single reaction mixture. In some embodiments, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 or more different labels may be used in a single reaction mixture or multiple reaction mixtures. Using such multiplexing techniques allows for independent monitoring of each target nucleic acid. In some embodiments, multiple target nucleic acids can be detected using a single labeled primer or probe. Heating curves can be generated to distinguish between two or more target nucleic acids each using the same label, but such analysis may not be necessary.

[0159] Polynucleotide modification

[0160] In some embodiments, the methods described herein for detecting at least one target nucleic acid employ one or more modified polynucleotides, such as polynucleotides containing one or more affinity-enhancing nucleotide analogs. Modified polynucleotides available in the methods described herein include primers for reverse transcription, PCR amplification primers, and probes. In some embodiments, the incorporation of affinity-enhancing nucleotides improves the binding affinity and specificity of the polynucleotide to its target nucleic acid compared to polynucleotides containing only canonical deoxyribonucleotides, allowing the use of shorter polynucleotides or allowing for shorter complementary regions between the polynucleotide and the target nucleic acid.

[0161] In some embodiments, the affinity-enhancing nucleotide analogs comprise nucleotides containing one or more base modifications, sugar modifications, and / or backbone modifications. In some embodiments, the modified bases used in the affinity-enhancing nucleotide analogs include 5-methylcytosine, isocytosine, pseudoisocytosine, 5-bromouracil, 5-propynyluracil, 6-aminopurine, 2-aminopurine, inosine, diaminopurine, 2-chloro-6-aminopurine, xanthine, and hypoxanthine. In some embodiments, the affinity-enhancing nucleotide analogs comprise nucleotides having modified sugars, such as 2'-substituted sugars, such as 2'-O-alkyl-ribose, 2'-amino-deoxyribose, 2'-fluoro-deoxyribose, 2'-fluoro-arabinose, and 2'-O-methoxyethyl-ribose (2'MOE). In some embodiments, the modified sugar is arabinose or d-arabinose-hexetol.

[0162] In some embodiments, affinity-enhancing nucleotide analogs include backbone modifications, such as the use of peptide nucleic acids (PNAs; for example, oligomers comprising nucleobases linked together by an amino acid backbone). Other backbone modifications include phosphate thioester linkages, phosphodiester-modified nucleic acids, combinations of phosphodiester and phosphate thioester nucleic acids, methylphosphonates, alkylphosphonates, phosphate esters, alkyl phosphate thioesters, aminophosphate esters, carbamates, carbonates, triphosphate esters, acetamidates, carboxymethyl esters, methyl phosphate thioesters, dithiophosphate esters, p-ethoxy modifications, and combinations thereof.

[0163] In some embodiments, the polynucleotide includes at least one affinity-enhanced nucleotide analog having modified bases, at least one nucleotide having modified sugars (which may be the same nucleotide), and / or at least one non-naturally occurring internucleotide bond.

[0164] In some embodiments, the affinity-enhancing nucleotide analogue comprises a locked nucleic acid (“LNA”) sugar, which is a bicyclic sugar. In some embodiments, the polynucleotide used in the methods described herein comprises one or more nucleotides having an LNA sugar. In some embodiments, the polynucleotide comprises one or more regions consisting of nucleotides having an LNA sugar. In other embodiments, the polynucleotide comprises nucleotides having an LNA sugar dispersed with deoxyribonucleotides. See, for example, Frieden, M. et al. (2008) Curr. Pharm. Des. 14(11):1138-1142.

[0165] Primers

[0166] In some implementations, the polynucleotide is the primer. Primers available in the methods described herein are generally capable of selectively hybridizing with: genomic DNA, target RNA (genomic or transcript), cDNA reverse-transcribed from target RNA, and / or amplicon amplified from genomic DNA, target RNA, or cDNA (collectively, the “template”), and can be extended to form a primer extension product in the presence of the template, polymerase, and suitable buffers and reagents. Primers are generally of sufficient length to ensure selective hybridization with their target nucleic acid. Typically, primers of at least 15 nucleotides in length hybridize specifically in most cases, and this length can be shortened, for example, by including affinity-enhancing modifications, such as those discussed above. Primers can, but do not necessarily, be perfectly complementary to their target nucleic acid. Primers can have any degree of complementarity described above for the exemplary polynucleotides. In some illustrative embodiments, primers may be: 8 to 40 nucleotides in length and at least 90% complementary to their target nucleic acid; 8 to 40 nucleotides in length and at least 95% complementary to their target nucleic acid; 8 to 40 nucleotides in length and at least 99% complementary to their target nucleic acid; 8 to 30 nucleotides in length and at least 90% complementary to their target nucleic acid; 8 to 30 nucleotides in length and at least 95% complementary to their target nucleic acid; or 8 to 30 nucleotides in length and at least 99% complementary to their target nucleic acid. In some embodiments where the primer is less than 100% complementary to its target nucleic acid, making the 3' nucleotide of the primer complementary to its target nucleic acid is beneficial for the generation of the extension product.

[0167] In some embodiments, under the same assay conditions, primers selectively hybridizing with their target nucleic acid exhibit at least 5-fold higher affinity for the target nucleic acid than for non-target nucleic acids. In some embodiments, under the same assay conditions, primers selectively hybridizing with their target nucleic acid exhibit at least 10-fold higher affinity for the target nucleic acid than for non-target nucleic acids.

[0168] In some implementations, primer pairs are designed to produce amplicons that are 50 to 1500 nucleotides long, 50 to 1000 nucleotides long, 50 to 750 nucleotides long, 50 to 500 nucleotides long, 50 to 400 nucleotides long, 50 to 300 nucleotides long, 50 to 200 nucleotides long, 50 to 150 nucleotides long, 100 to 300 nucleotides long, 100 to 200 nucleotides long, or 100 to 150 nucleotides long.

[0169] In some implementations, the primers are labeled with a detectable portion. In some implementations, the primers are not labeled.

[0170] probe

[0171] In some implementations, the polynucleotide is the probe. Probes available in the methods described herein are generally capable of selectively hybridizing with: genomic DNA, target RNA (genomic or transcript), cDNA reverse-transcribed from target RNA, and / or amplicons amplified from genomic DNA, target RNA, or cDNA (collectively, the “template”). Typically, probes of at least 15 nucleotides in length hybridize specifically in most cases, and this length can be shortened, for example, by including affinity-enhancing modifications, such as those discussed above. The probe can, but does not necessarily, be perfectly complementary to its target nucleic acid. The probe can have any degree of complementarity described above for the exemplary polynucleotides. In some illustrative embodiments, the probe may be: 8 to 40 nucleotides in length and at least 90% complementary to its target nucleic acid; 8 to 40 nucleotides in length and at least 95% complementary to its target nucleic acid; 8 to 40 nucleotides in length and at least 99% complementary to its target nucleic acid; 8 to 30 nucleotides in length and at least 90% complementary to its target nucleic acid; 8 to 30 nucleotides in length and at least 95% complementary to its target nucleic acid; or 8 to 30 nucleotides in length and at least 99% complementary to its target nucleic acid. In some embodiments where the primer is less than 100% complementary to the target nucleic acid, any non-complementary sites or regions are typically configured such that the probe's ability to selectively hybridize with its target nucleic acid is not impaired.

[0172] In some embodiments, under the same assay conditions, a probe selectively hybridizing with its target nucleic acid exhibits at least 5 times higher affinity for the target nucleic acid than for non-target nucleic acids. In some embodiments, under the same assay conditions, a probe selectively hybridizing with its target nucleic acid exhibits at least 10 times higher affinity for the target nucleic acid than for non-target nucleic acids.

[0173] Polynucleotide markers

[0174] In some embodiments, primers or probes are labeled with a detectable portion. The detectable portion includes a directly detectable portion (e.g., a fluorescent dye) and an indirectly detectable portion (e.g., a member of a binding pair). When the detectable portion is a member of a binding pair, in some embodiments, the probe can be detected by incubating it with a detectable label bound to a second member of the binding pair. In some embodiments, primers or probes are unlabeled, for example, when they are immobilized on, for example, microarrays or beads. Labeled primers are extendable, for example, by polymerase. In some embodiments, probes are extendable. In other embodiments, probes are non-extendable. The following discussion focuses on probes, as these probes are more commonly used for detection in the methods described herein; however, those skilled in the art will understand that the polynucleotide labeling strategies described below are equally applicable to primer labeling.

[0175] In some embodiments, the probe is a FRET probe, which is labeled with a fluorescent dye (donor) at its 5' end and with a quencher (acceptor) at its 3' end. The quencher is a chemical group that absorbs (i.e., inhibits) fluorescence emission from the dye when the groups are very close (e.g., attached to the same probe). Therefore, in some embodiments, the emission spectrum of the dye should overlap considerably with the absorption spectrum of the quencher. In other embodiments, the dye and quencher are not at the ends of the FRET probe.

[0176] Exemplary FRET probes include, but are not limited to, TaqMan® probes, molecular beacon probes, and Scorpion probes. TaqMan® probes are linear probes that typically have a fluorescent dye covalently bound to one end of DNA and a quencher molecule covalently bound to another location (e.g., the other end of DNA). FRET probes contain a sequence complementary to a region of cDNA or amplicon, such that when the FRET probe hybridizes with cDNA or amplicon, the dye fluorescence is quenched, and when the probe is digested during amplification of the cDNA or amplicon, the dye is released from the probe and generates a fluorescent signal. In some embodiments, the amount of target nucleic acid in the sample is proportional to the amount of fluorescence measured during amplification.

[0177] Similar to TaqMan® probes, molecular beacons use FRET to detect PCR products via probes with a fluorescent dye and quencher attached to their ends. Unlike TaqMan® probes, molecular beacons remain intact during PCR cycles. When free in solution, the molecular beacon probe forms a stem-loop structure, allowing the dye and quencher to come close enough to induce fluorescence quenching. When the molecular beacon hybridizes with the target nucleic acid, the stem-loop structure is eliminated, causing the dye and quencher to spatially separate, and the dye fluoresces. Molecular beacons can be obtained from, for example, GeneLink. TM(See www.genelink.com / newsite / products / mbintro.asp for details.)

[0178] In some implementations, Scorpion probes can be used as sequence-specific primers and for PCR product detection. Like molecular beacons, Scorpion probes form a stem-loop structure when not hybridizing with the target nucleic acid. However, unlike molecular beacons, Scorpion probes achieve both sequence-specific priming and PCR product detection. A fluorescent dye molecule is attached to the 5' end of the Scorpion probe, and a quencher is attached at another location, such as the 3' end. The 3' portion of the probe is complementary to the extension product of the PCR primer, and this complementary portion is linked to the 5' end of the probe via a non-amplifiable portion. After Scorpion primer extension, the target-specific sequence of the probe binds to the complementary sequence within the extended amplicon, thereby opening the stem-loop structure and allowing the dye at the 5' end to fluoresce and generate a signal. Scorpion probes are available from, for example, Premier Biosoft International (see www.premierbiosoft.com / tech_notes / Scorpion.html).

[0179] In some implementations, labels that can be used on FRET probes include colorimetric dyes and fluorescent dyes, such as Alexa Fluor dyes; BODIPY dyes, such as BODIPY FL, Cascade Blue, and Cascade Yellow; coumarins and their derivatives, such as 7-amino-4-methylcoumarin, aminocoumarin, and hydroxycoumarin; anthocyanin dyes, such as Cy3 and Cy5; eosin and erythrosine; fluorescein and its derivatives, such as fluorescein isothiocyanate; and macrocyclic chelates of lanthanides, such as Quantum Dye. TM Marina Blue; Oregon Green; Rhodamine dyes, such as Rhodamine Red, Tetramethylrhodamine and Rhodamine 6G; Texas Red; Fluorescent energy transfer dyes, such as Thiazole Orange-Ethidium Heteromer; and TOTAB.

[0180] Specific examples of dyes include, but are not limited to, those identified above and the following: Rhodamine Green, Rhodamine Red, renal contrast agent (Renographin), ROX, SYPRO, TAMRA, 2',4',5',7'-Tetrabromosulfonefluorescein, and TET.

[0181] Examples of dye / quencher pairs (i.e., donor / acceptor pairs) include, but are not limited to: fluorescein / tetramethylrhodamine; IAEDANS / fluorescein; EDANS / dabcyl; fluorescein / fluorescein; BODIPY FL / BODIPY FL; and fluorescein / QSY 7 or QSY 9 dyes. When the donor and acceptor are the same, in some embodiments, FRET can be detected by fluorescence depolarization. Specific examples of dye / quencher pairs (i.e., donor / acceptor pairs) include, but are not limited to: In some cases, the same quencher can be used for multiple dyes, for example, broad-spectrum quenchers such as Iowa Black® quencher (Integrated DNA Technologies, Coralville, IA) or Black Hole Quencher. TM (BHQ) TM ; Sigma-Aldrich, St. Louis, MO).

[0182] Specific examples of fluorescently labeled ribonucleotides that can be used to prepare probes for some embodiments of the methods described herein are available from Molecular Probes (Invitrogen), and these include Alexa Fluor488-5-UTP, fluorescein-12-UTP, BODIPY FL-14-UTP, BODIPY TMR-14-UTP, tetramethylrhodamine-6-UTP, Alexa Fluor 546-14-UTP, Texas Red-5-UTP, and BODIPY TR-14-UTP. Other fluorescent ribonucleotides are available from Amersham Biosciences (GE Healthcare), such as Cy3-UTP and Cy5-UTP.

[0183] Specific examples of fluorescently labeled deoxyribonucleotides that can be used to prepare probes for the methods described herein include dinitrophenyl (DNP)-1'-dUTP, Cascade Blue-7-dUTP, Alexa Fluor 488-5-dUTP, fluorescein-12-dUTP, Oregon Green 488-5-dUTP, BODIPY FL-14-dUTP, Rhodamine Green-5-dUTP, Alexa Fluor 532-5-dUTP, BODIPY TMR-14-dUTP, tetramethylrhodamine-6-dUTP, Alexa Fluor 546-14-dUTP, Alexa Fluor 568-5-dUTP, Texas Red-12-dUTP, Texas Red-5-dUTP, BODIPY TR-14-dUTP, Alexa Fluor 594-5-dUTP, and BODIPY... 630 / 650-14-dUTP, BODIPY 650 / 665-14-dUTP, Alexa Fluor 488-7-OBEA-dCTP, Alexa Fluor 546-16-OBEA-dCTP, Alexa Fluor 594-7-OBEA-dCTP, and Alexa Fluor 647-12-OBEA-dCTP. The fluorescently labeled nucleotides are commercially available and can be purchased from, for example, Invitrogen.

[0184] As described above, the exemplary detectable portion also includes members of a binding pair. Exemplary binding pairs include, but are not limited to, biotin and streptavidin, antibodies and antigens, etc.

[0185] A single PCR reaction (nucleic acid amplification) or multiple PCR reactions (nucleic acid amplification and / or denaturation analysis) running sequentially (or simultaneously in separate temperature-controlled channels or chambers) can use the same detectable label because the PCR signal DNA running sequentially is analyzed sequentially, and simultaneous PCR signal DNA is distinguished by its presence in different temperature-controlled channels or chambers. The signal generated by this amplification can be distinguished from other amplification products because it was not run simultaneously and / or because it was run in different reaction channels / chambers. However, when multiple nucleic acid amplifications are run simultaneously in the same chamber, the reaction products analyzed each time are typically detected and / or quantified using different and distinguishable labels.

[0186] In some embodiments, the amplification products (amplified nucleic acids from a nucleic acid assay) can be detected using methods well-known to those skilled in the art. In some embodiments, amplification is a direct and simple PCR amplification reaction. However, in some embodiments, nested PCR reactions are used to amplify nucleic acids from a nucleic acid assay. In several embodiments, multiplex PCR assays are considered, particularly when it is desirable to analyze multiple products of a nucleic acid assay in the same amplification reaction. In some embodiments, in such multiplex amplification reactions, each probe (e.g., for each specific analyte) has its own specific dye / fluor, making it detectable independently of other probes. In some embodiments, generally, to generate a signal, probes used in multiple amplification reactions utilize fluorescence changes of a fluorophore, which is due to changes in the interaction between the fluorophore and another molecule or moiety interacting with it, caused by altering the distance between the fluorophore and the interacting molecule or moiety used to detect and / or quantify the amplification product. Alternatively, other methods for detecting polynucleotides in a sample are considered, including but not limited to the use of radiolabeled probes.

[0187] sample

[0188] The sample to be tested can be any sample suspected of containing the target nucleic acid. In some embodiments, the sample is a biological sample collected from the object. In other embodiments, the sample is a sample not collected directly from the object, such as a wastewater sample or a sample from an air filter in a building.

[0189] Exemplary biological samples include the following: sputum samples, nasal aspirate samples, nasal wash samples, nasal swab samples, nasopharyngeal swab samples, saliva samples, oropharyngeal swab samples, pharyngeal swab samples, bronchoalveolar lavage samples, bronchial aspirate samples, bronchial wash samples, tracheal aspirate samples, tracheal wash samples, tracheal aspirate samples, nasal secretion samples, mucus samples, pleural effusion samples, cerebrospinal fluid samples, fecal samples, tissue biopsy samples, respiratory samples, or combinations thereof.

[0190] In some embodiments, the sample to be tested is fresh (i.e., never frozen). In other embodiments, the sample is a frozen sample. In some embodiments, the sample is a tissue sample, such as a formalin-fixed paraffin-embedded sample. In some embodiments, the sample is a liquid cytology sample. In some embodiments, for example, in the determination of tuberculosis drug resistance, the sample may be an untreated or purified, digested, and concentrated sputum sample.

[0191] In some embodiments, the sample to be tested is contacted with a buffer after collection. For example, in the case of a sputum sample, a buffer (including, for example, a preservative) may be added to the sample. In some embodiments where the sample is a swab sample, the swab may simply be placed in the buffer. In some embodiments, the sample is contacted with the buffer immediately; in the case of a swab, the swab is placed in the buffer immediately. In some embodiments, the sample (e.g., including a swab) is contacted with the buffer within 5 minutes, 10 minutes, 30 minutes, 1 hour, or 2 hours of sample collection.

[0192] In some embodiments, the method of the present invention uses a sample or buffer sample of less than 5 ml, less than 4 ml, less than 3 ml, less than 2 ml, less than 1 ml, or less than 0.75 ml. In some embodiments, the method of the present invention uses a sample or buffer sample of 0.1 ml to 1 ml.

[0193] object

[0194] Biological samples that can be used in the methods described herein can be collected from any object that may have one or more target nucleic acids. In various embodiments, the object may include non-human animals, such as canines, felines, equines, primates and other non-human mammals, as well as humans.

[0195] In some implementations, the samples to be tested are obtained from individuals who have one or more symptoms of tuberculosis or who have been exposed to such individuals. The main symptoms of tuberculosis include persistent cough, cough with blood in sputum, fever, and chest pain.

[0196] In some embodiments, the methods described herein can be used for routine screening of seemingly healthy individuals without risk factors. In some embodiments, the methods described herein are used to screen asymptomatic individuals, such as during routine or preventative care. In some embodiments, the methods described herein are used to screen women who are pregnant or attempting to become pregnant.

[0197] In some implementations, the methods described herein can be used to assess the effectiveness of treatment in an individual receiving treatment (e.g., tuberculosis).

[0198] Determination methods

[0199] Any analytical procedure capable of specifically detecting the target nucleic acid can be used in the methods presented herein. In some embodiments, the DNA target can be detected by direct hybridization or, more readily, by amplification of the DNA template and detection of the amplicon. In some embodiments, the RNA target can be detected by direct hybridization or, more readily, by reverse transcription of the target RNA to generate cDNA complementary to the target RNA. This cDNA can be detected directly by direct hybridization or by amplification of the cDNA template.

[0200] Nucleic acid amplification provides rapid, sensitive, and specific detection of nucleic acid targets and has been used in a wide variety of assays to detect nucleic acid targets. Following the guidance herein, those skilled in the art can implement the methods described herein in any number of different nucleic acid amplification-based assays, such as using any of the nucleic acid amplification methods discussed above. Such methods may require thermal cycling, but do not necessarily, as in the case of isothermal amplification. Exemplary methods include, but are not limited to, isothermal amplification, real-time RT-PCR, endpoint RT-PCR, and amplification using T7 polymerase from a T7 promoter annealed with DNA, such as SenseAmp Plus, available from Implen, Germany. TM The kit is provided. Amplification and detection can be performed in solution or using a solid support (e.g., a biochip). Nucleic acid amplification-based assays can be performed in a single reaction chamber or multiple reaction chambers. Amplification can be nested or non-nested. In some embodiments, detection includes electrochemical detection.

[0201] In some embodiments, the target nucleic acid and / or optional controls can be detected by: (a) contacting a nucleic acid from the sample with a set of primers and optional probes to detect the presence of the desired target nucleic acid; (b) subjecting the nucleic acid, primers, and optional probes to amplification conditions; (c) detecting the presence of any amplification products, optionally by real-time PCR, melting curve analysis, or a combination thereof; and (d) differentiating the presence of viral pathogens in the sample, or determining the absence of viral pathogens detectable using the primer set, based on the detection or absence of amplification products, respectively. In this context, "differential identification" refers to the ability to determine the presence of a specific target organism being measured and the absence of one or more other target organisms. In some embodiments, the assay is capable of determining the presence of any target organism present in the sample while excluding the presence of other target organisms (above the detection limit of the assay).

[0202] In some embodiments of amplification via polymerase chain reaction (PCR), exemplary cycles include an initial denaturation at 90°C to 100°C for 20 seconds to 5 minutes, followed by a cycle of denaturation at 90°C to 100°C for 1 to 10 seconds, followed by annealing and amplification at 60°C to 75°C for 10 to 40 seconds. Another exemplary cycle includes up to three cycles of 20 seconds at 94°C, followed by up to three cycles of 1 second at 95°C and 35 seconds at 62°C; 20 cycles of 1 second at 95°C and 20 seconds at 62°C; and 14 cycles of 1 second at 95°C and 35 seconds at 62°C. In some embodiments, the denaturation step is omitted for the first cycle after the initial denaturation step. In some embodiments, Taq polymerase is used for amplification. In some embodiments, the cycles are performed at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, or at least 45 times. In some implementations, Taq is used in conjunction with a hot-start function. In some implementations, detection of the target nucleic acid occurs within less than 3 hours, less than 2.5 hours, less than 2 hours, less than 1 hour, or less than 30 minutes from initial denaturation to the final extension. In some implementations, the target nucleic acid is detected by methods including real-time quantitative PCR, for example, using FRET probes, such as those described above.

[0203] In some embodiments, real-time PCR results are quantified by constructing a standard curve from known concentrations of nucleic acids and then extrapolating the quantification information of unknown concentrations of target nucleic acids. In some embodiments, the nucleic acid used to generate the standard curve is DNA (e.g., an endogenous or exogenous control). In some embodiments, the nucleic acid used to generate the standard curve is purified double-stranded plasmid DNA or in vitro-generated single-stranded DNA.

[0204] In some embodiments, for an assay to indicate the absence of a given target nucleic acid in a sample, the Ct values ​​for endogenous controls (e.g., SAC) and / or exogenous controls (e.g., SPC) must be within a previously determined valid range. For example, in some embodiments, the absence of a specific target nucleic acid cannot be confirmed unless a control is detected, indicating a successful assay.

[0205] In some implementations, a threshold Ct (or “cutoff Ct”) value for the target nucleic acid (including endogenous and / or exogenous controls) has been previously determined, below which the gene is considered to be detected. In some implementations, the threshold Ct is determined using substantially the same assay conditions and system (e.g., GENEXPERT®) as the sample to be tested thereon.

[0206] Real-time PCR can be performed using any PCR instrument available in the art. Typically, instruments used for real-time PCR data collection and analysis include a thermal cycler, optics for fluorescence excitation and emission collection, and optionally a computer and data acquisition and analysis software.

[0207] In some implementations, the number of target nucleic acids in the assay exceeds, for example, the number of markers that can be detected in a particular instrument. Therefore, a melt-down assay can be performed after PCR amplification to increase the number of results that can be reported. Generally, target organisms requiring high sensitivity can be detected by real-time PCR using TaqMan probes or molecular beacon probes, and / or by melt-down assays.

[0208] Another method for detecting target nucleic acids may include separate high-resolution melting. Endpoint melting curve data can be obtained to detect and analyze the target nucleic acid, producing results for each analyte.

[0209] Target nucleic acids can also be detected by real-time PCR, but in more than one reaction chamber. Another method for detecting target nucleic acids can include digital microfluidics or electrowetting and electrochemical detection. For example, digital microfluidics or electrowetting can be used to control the movement and transfer of samples and reagents within the cartridge. Such a system can include a microarray for detection, consisting of target-specific capture probes attached to a gold electrode (solid support), which generates a voltage signal if the "target DNA / signal probe" hybridizes with the capture probe. Target nucleic acids can also be detected using chips that include integrated sensor arrays.

[0210] Some examples of other methods that can be used with the methods described herein include bead-based flow cytometry assays. See Lu J. et al. (2005) Nature 435:834-838, whose description of that is incorporated herein by reference. An example of bead-based flow cytometry assays is the xMAP® technology of Luminex, Inc. See www.luminexcorp.com / technology / index.html. Another method uses microfluidic devices and single-molecule detection. See Fuchs et al., U.S. Patent Nos. 7,402,422 and 7,351,538, US Genomics, Inc., each of which is incorporated herein by reference in its entirety. Another method is simple gel electrophoresis and detection with labeled probes (e.g., probes labeled with radioactive or chemiluminescent labels), such as detection via northern blotting.

[0211] In some implementations, methods for detecting target nucleic acids do not include bead-based flow cytometry assays, microfluidic devices and single-molecule detection, simple gel electrophoresis, use of capture probes attached to a solid support, separation of the reaction mixture into multiple reaction chambers, array-based detection, nested amplification, electrochemical detection, high-resolution melting only, or combinations thereof.

[0212] Automated measurement methods

[0213] Easy-to-automate methods are of great interest. The methods described herein can be performed in a substantially automated manner using commercially available nucleic acid amplification systems. Exemplary non-limiting nucleic acid amplification systems that can be used to implement the methods of the present invention include the GENEXPERT® system, the GENEXPERT® Infinity system, and the GENEXPERT® Xpress system (Cepheid, Sunnyvale, Calif.). In some embodiments, the amplification system can be available at the same location as the individual being tested, such as the office, clinic, or community hospital of a healthcare provider, so that processing is not delayed by transporting the sample to another facility. Using an automated system, such as the GENEXPERT® system, assays according to the methods described herein can be completed within 3 hours, in some embodiments within 2 hours, in some embodiments within 1 hour, in some embodiments within 45 minutes, in some embodiments within 35 minutes, and in some embodiments within 30 minutes. GENEXPERT® uses individual, disposable cartridges. Sample extraction, amplification, and detection can all be performed within this individual cartridge as described herein.

[0214] In some embodiments, after the sample is added to the cartridge, the sample is contacted with a lysis buffer, and the released nucleic acid (NA) binds to a NA-binding matrix, such as silica or a glass matrix. The sample supernatant is then removed, and the NA is eluted in an elution buffer (e.g., Tris / EDTA buffer). The eluent can then be processed within the cartridge to detect the target nucleic acid as described herein. In some embodiments, the eluent is used to reconstruct at least some PCR reagents, which are present in the cartridge as lyophilized particles.

[0215] A multi-chamber cartridge may contain the primer set described herein and optional probes or subsets thereof disposed within the chambers. In some embodiments, the primer set described herein and optional probes or subsets thereof are disposed in more than one of the multiple chambers.

[0216] In some embodiments, RT-PCR is used to amplify the target nucleic acid and analyze its presence. In some embodiments, reverse transcription is performed using MMLV and / or CAT-A RT enzymes, incubated at 40°C to 50°C for 5 to 20 minutes. In some embodiments, PCR is performed using a Taq polymerase with hot-start capability, such as AptaTaq (Roche). In some embodiments, initial denaturation is performed at 90°C to 100°C for 20 seconds to 5 minutes; cyclic denaturation is performed at 90°C to 100°C for 1 to 10 seconds; cyclic annealing and amplification are performed at 60°C to 75°C for 10 to 40 seconds; and up to 50 cycles are performed.

[0217] In some embodiments, a double-denaturation method is used to amplify low-copy-number target nucleic acids. In some embodiments, the double-denaturation method includes a first denaturation step followed by the addition of primers and / or probes for detecting the target nucleic acid. The sample containing all or most of the nucleic acid (e.g., DNA elution buffer) is then subjected to a second denaturation, and in some cases, a portion of the sample is aliquoted for cycling and detection of the target nucleic acid. While not intended to be bound by any particular theory, the double-denaturation protocol increases the chance of low-copy-number target nucleic acids (or their complements) being present in the aliquots selected for cycling and detection because the second denaturation effectively doubles the number of target nucleic acids before the aliquots are selected for cycling (i.e., it separates the target nucleic acid and its complement into two separate templates). In some embodiments, the first denaturation step includes heating to a temperature of 90°C to 100°C for a total time of 30 seconds to 5 minutes. In some embodiments, the second denaturation step includes heating to a temperature of 90°C to 100°C for a total time of 5 seconds to 3 minutes. In some embodiments, the first and / or second denaturation steps are performed by separately heating aliquots of the sample. In some embodiments, each aliquot may be heated for the durations listed above. As a non-limiting example, a first denaturing step for a NA-containing sample (e.g., DNA elution buffer) may include heating at least one, at least two, at least three, or at least four aliquots of the sample individually (sequentially or simultaneously) to a temperature of 90°C to 100°C for 60 seconds each time. As a non-limiting example, a second denaturing step for a NA-containing sample (e.g., DNA elution buffer) containing enzymes, primers, and probes may include heating at least one, at least two, at least three, or at least four aliquots of the elution buffer individually (sequentially or simultaneously) to a temperature of 90°C to 100°C for 5 seconds each time. In some embodiments, the aliquots are whole NA-containing samples (e.g., DNA elution buffer). In some embodiments, the aliquots are less than the whole NA-containing sample (e.g., DNA elution buffer).

[0218] In some implementations, for example, offline centrifugation is used for samples with low cell content. The sample, with or without added buffer, is centrifuged and the supernatant is removed. The precipitate is then resuspended in a smaller volume of supernatant or buffer. The resuspended precipitate is then analyzed as described herein.

[0219] Exemplary Automation and Systems

[0220] Many existing fully integrated nucleic acid amplification and assay systems capable of sample preparation are often quite complex and expensive. The nucleic acid amplification and assay system presented in this article provides rapid, simple, convenient, and affordable nucleic acid analysis.

[0221] System Overview

[0222] In one aspect, the present invention relates to a sample cartridge utilizing a valve body platform that allows for the detection of enveloped and free target nucleic acids. In some embodiments, the valve body includes a sample processing area or lysis chamber providing either or both of mechanical and chemical lysis. This allows a single cartridge to provide lysis for multiple different types of targets and can therefore be considered a “setup cartridge.” In some embodiments, the sample cartridge can perform the processing and detection of viral targets suitable for chemical lysis.

[0223] The sample cartridge device can be any device configured to perform one or more process steps associated with the preparation and / or analysis of biological fluid samples according to any of the methods described herein. In some embodiments, the sample cartridge device is configured to perform at least sample preparation. The sample cartridge can also be configured to perform additional processes, such as detecting target nucleic acids in a nucleic acid amplification test (NAAT) (e.g., polymerase chain reaction (PCR) assay), by using a reaction vessel connected to the sample cartridge. In some embodiments, the reaction vessel extends from the cartridge body. The preparation of fluid samples typically involves a series of processing steps, which, depending on the specific protocol, may include chemical, electrical, mechanical, thermal, optical, or acoustic processing steps. Such steps can be used to perform a variety of sample preparation functions, such as cell capture, cell lysis, analyte binding, and binding of undesirable substances.

[0224] Sample cartridges suitable for use with the present invention include one or more transfer interfaces through which prepared fluid samples can be transferred to a connected reaction vessel for analysis. Figure 1A illustrates an exemplary assay cartridge 100 according to some embodiments, which, when received in an instrument module, is suitable for sample preparation and analytical testing via PCR. The sample cartridge is connected to a reaction vessel 116 (also referred to as a “reaction tube” or “PCR tube”) adapted to analyze fluid samples processed within the sample cartridge 100. In some embodiments, the reaction vessel extends from the cartridge body. Such a sample cartridge 100 includes multiple components including a main housing 102 having one or more chambers 108 for processing fluid samples, typically including sample preparation prior to analysis. In these embodiments, the sample cartridge may be a fully integrated nucleic acid amplification and testing system combining sample preparation, amplification, and detection. The instrument module facilitates the execution of the processing steps required for sample preparation, and the prepared sample is delivered via one of a pair of transfer interfaces to a fluid conduit in the reaction vessel 116 connected to the housing of the sample cartridge 100. The prepared biofluid sample is then delivered to the reaction chamber of the reaction vessel, where it undergoes nucleic acid amplification. In some embodiments, the amplification is a polymerase chain reaction. In some embodiments, concurrent with the amplification of the biofluid sample, the excitation and optical detection devices of the module are used to detect light emission indicating the presence or absence of a target nucleic acid analyte (e.g., bacteria, viruses, pathogens, toxins, or other target analytes). It should be understood that such a reaction vessel may include a variety of different chambers, conduits, or micropore arrays for detecting the target analyte. The sample cartridge may be provided with means for preparing the biofluid sample prior to delivery to the reaction vessel. Any chemical reagents required for virus or cell lysis, or devices for binding or detecting the target analyte (e.g., reagent beads), may be contained within one or more chambers of the sample cartridge and thus may be used for sample preparation.

[0225] Exemplary uses of a reaction vessel for analyzing biological fluid samples are described in commonly assigned U.S. Patent No. 6,818,185, filed May 30, 2000, entitled “Cartridge for Conducting a Chemical Reaction,” the entire contents of which are incorporated herein by reference for all purposes. Examples of sample boxes and related modules are shown and described in U.S. Patent No. 6,374,684, filed August 25, 2000, entitled "Fluid Control and Processing System"; U.S. Patent No. 8,048,386, filed February 25, 2002, entitled "Fluid Processing and Control"; U.S. Patent Application No. 63 / 217,672, filed July 1, 2021, entitled "Universal Assay Cartridge and Methods of Use"; U.S. Provisional Application No. 63 / 319,993, filed March 15, 2022, entitled "Unitary Cartridge Body and Associated Components and Methods of Manufacture"; and U.S. Patent No. 63 / 219,993, filed July 22, 2016, entitled "Molecular Diagnostic Assay System". 10,562,030; its entire contents are incorporated herein by reference for all purposes.

[0226] Several aspects of the sample cartridge 100 can be further understood by referring to U.S. Patent No. 6,374,684 (“684 Patent”), which describes certain aspects of the sample cartridge in more detail. Such a sample cartridge may include a fluid control mechanism, such as a rotary fluid control valve assembly, fluidly connected to a chamber of the sample cartridge. The term “chamber” is used interchangeably with the terms “orifice,” “tube,” etc. Rotation of the rotary fluid control valve allows fluid communication between the chamber and the valve to control the flow of a biological fluid sample deposited in the cartridge into different chambers, in which various reagents can be supplied as needed according to a specific protocol to prepare a biological fluid sample for analysis. To operate the rotary valve, the cartridge processing module includes a motor (e.g., a stepper motor), which is typically coupled to a drive system that is characteristically engaged with the valve in the sample cartridge to control the movement of the valve in coordination with the movement of the syringe, thereby causing the fluid sample to move according to a desired sample preparation protocol. The “684 Patent” illustrates the fluid metering and dispensing function of the rotary valve according to a specific sample preparation protocol.

[0227] Exemplary measuring kit

[0228] As shown in Figure 1A, the assay cartridge 100 includes a housing 102 containing a plurality of chambers 108 for reagent or buffer preparation and sample handling. These chambers are arranged around a central syringe barrel 106, which is in fluid communication with a valve body 110 (see Figure 1A). Figure 1B and 1C The valve body 110 may include a cap 112, and the entire housing may be supported on a housing base 101. The valve body typically includes a channel or cavity (chamber 114) that may contain a filter as described herein, which can be used for binding and eluting nucleic acids. In some embodiments, the housing may also include one or more temperature-controlled channels or chambers, which may serve as thermal cycling chambers in some embodiments. A "plunger" (not shown) is operable to draw fluid into a syringe barrel 106, and rotation of the valve body 110 provides selective fluid communication between the plurality of reagent chambers 108 and channels, reaction chambers, mixing chambers, and optionally any temperature-controlled areas. Thus, the plurality of reagent chambers 108, reaction chambers, filter material, and temperature-controlled chambers or channels are selectively fluidly communicated by rotation of the plunger, and reagent movement (e.g., chamber loading or unloading) is operated by the "syringe" action of the plunger within the valve assembly. In other embodiments, multiple reagent chambers, reaction chambers, filter materials, and temperature-controlled chambers or channels are selectively fluidly connected by the linear movement of reagents and samples from one chamber to the next (e.g., by forced motion).

[0229] Reaction Module

[0230] In some implementations, the box 100 is configured to be inserted into the reaction module 300, for example, as Figure 2A As shown. Figure 2B As shown, the module is configured to receive the cartridge 100 therein. In some embodiments, the reaction module provides a heating plate 308 to heat the temperature-controlled chamber or channel. The module may optionally additionally include a fan 304 to provide cooling if the temperature-controlled channel or chamber is a heat-circulating channel or chamber. Electronic circuitry 302 may be provided to transmit information (e.g., optical information) to a computer for analysis. In some embodiments, the module may include an optical block 306 to provide the excitation and / or detection of one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) optical signals representing signal DNA amplified, for example, against a variety of PCR targets. In several embodiments, an electrical connector 312 may be provided for connecting the module to a system (e.g., a system controller, or to a separate analysis / controller unit). As shown, in Figure 2B In this process, a pipette 310 can be used to introduce the sample into the cartridge. In some embodiments, the module also includes a controller that operates the plunger in the syringe barrel and the rotation of the valve body.

[0231] While the methods described herein are primarily referred to in connection with the GENEXPERT® cartridge of Cepheid Inc. (Sunnyvale, Calif.) (see, for example, Figure 1A), it should be recognized that, in light of the teachings provided herein, these methods can be implemented on other cartridge / microfluidic systems, including alternative cartridge designs with valve assemblies involving multiple interface components, and cartridge bodies with multiple chambers defined by multiple interface components to form a cartridge (e.g., those described in Korean Applications No. 102293717B1 and KR102362853B1), cartridges that utilize ultrasound to lyse cells in biological samples (e.g., those described in International Application No. WO2021 / 245390A1), and cartridges and systems that utilize electrowetting grids for microdroplet manipulation and arrays of electrical sensors configured to detect the target analyte (e.g., in International Application No. WO2021 / 245390A1). These include cartridges that facilitate the movement of nucleic acids from one chamber to the next by opening a venting bag (e.g., those described in International Application No. WO2012 / 145730A2), multiplex assay systems that include multiple thermal cycling units, allowing individual chambers to be heated, cooled, and / or compressed to mix fluids within the chambers or to propel fluids from one chamber to another (e.g., those described in International Application No. WO2015 / 138343A1), and systems for rapid nucleic acid amplification that facilitate nucleic acid amplification by arranging flexible portions of the sample cartridge to achieve temperature cycling (e.g., those described in International Application No. WO2017 / 147085A1). Such cartridge / microfluidic systems can include, for example, microfluidic systems implemented using soft lithography, microfluidic systems fabricated in micrometers / nanometers using hard lithography, etc.

[0232] In one exemplary embodiment, the cartridge may include multiple reaction chambers; more specifically, the reaction container may include multiple reaction chambers. In these embodiments, different types of lyophilized primers and probes may be provided in each reaction chamber. For example, primers and probes for virus-associated nucleic acids may be provided in one reaction chamber, and primers and probes for virus-associated nucleic acids may be provided in a second chamber for amplification and detection, etc. Of course, multiple amplification and detection processes can be performed simultaneously in a single reaction chamber. Therefore, the amplification of each target nucleic acid described herein can be performed individually in a separate reaction chamber or well, or in a multiplex reaction within a single reaction chamber or well.

[0233] Furthermore, it should be understood that the group assay methods described herein (i.e., identification based on multiple conditions of comparative levels of multi-target nucleic acids obtained from a single sample) can be further implemented in entirely different systems, including: gradient PCR, isothermal nucleic acid amplification systems, digital RT-PCR, electrochemical PCR, lateral flow assay kits, electrochemical sensors, nucleic acid sequencing, CRISPR / Cas-based technologies, chemiluminescence, and nanoparticle-based colorimetric detection.

[0234] In several implementations, signal DNA from a PCR (nucleic acid amplification) reaction is amplified for detection and / or quantification. In some implementations, amplification includes any of a variety of methods, including but not limited to polymerase chain reaction (PCR), ligase chain reaction (LCR), ligase detection reaction (LDR), multiple ligation-dependent probe amplification (MLPA), post-ligation Q-replicaase amplification, primer extension, strand displacement amplification (SDA), hyperbranched strand displacement amplification, multiple displacement amplification (MDA), nucleic acid strand-based amplification (NASBA), rolling circle amplification (RCA), etc.

[0235] Measurement Workflow

[0236] Prior to amplification, the sample undergoes one or more sample preparation steps. Typically, these steps include operations such as extracting intracellular material (e.g., nucleic acids) from whole-cell samples, viruses, etc., to form a crude extract for further processing of the sample for subsequent operations, such as denaturation, purification, filtration, desalting, etc., of contaminating (e.g., DNA-bound) proteins. The release of nucleic acids from sample cells or viruses and the denaturation of DNA-bound proteins can typically be performed using chemical, physical, or electrolytic lysis methods. For example, chemical methods typically use a lysis agent to rupture cells and extract nucleic acids, followed by treatment of the extract with a dissociative salt such as guanidine isothiocyanate or urea to denature any contaminating and potentially interfering proteins. Typically, when using chemical extraction and / or denaturation methods, suitable reagents can be incorporated into the sample preparation chamber, a separate accessible chamber, or introduced externally. Preferably, sample preparation is performed in only one or no more than two steps. For example, sample preparation may include heating the sample in a lysis solution without further purification prior to amplification. In some embodiments, the lysed sample may be diluted prior to amplification. One or more of these various sample preparation operations can be easily incorporated into the fluid-closed box system envisioned in this paper.

[0237] In one aspect, the assay kits described herein are capable of performing specific workflows that execute the lysis and detection of different target analytes as needed for a particular assay. In some embodiments, the kit is configured for the chemical lysis of multiple target organisms. In other embodiments, the kit is configured for the mechanical lysis of multiple target organisms. In still other embodiments, the kit is configured for both mechanical and chemical lysis to allow the lysis of multiple targets of different types. Thus, the kit can be configured to perform assays via existing workflows associated with conventional kits, or it can be operated according to new workflows specifically configured for assays.

[0238] In one aspect, it has as described in this article Figure 3 The sample cartridge of the valve assembly described in D is capable of performing multiple workflows, namely: chemical lysis of the target organism, mechanical lysis of the target organism, or both. Therefore, the sample cartridge can perform existing workflows associated with conventional cartridges, or it can perform entirely new workflows that combine both.

[0239] Exemplary assay workflows A through C (hereinafter) can be performed using a single universal cartridge. In any of these embodiments, the filter may be formed of a glass filter to facilitate affinity binding of nucleic acids (NA) to the glass fiber, and the pore size may also be suitable for chemical lysis. In any of these workflows, nucleic acid amplification may be PCR, real-time PCR, isothermal amplification (including but not limited to nucleic acid sequence-based amplification, loop-mediated isothermal amplification, helicase-dependent amplification, rolling circle amplification, multiple displacement amplification, whole genome amplification, or recombinase polymerase amplification), or other nucleic acid amplification methods known to those skilled in the art.

[0240] In workflow A, the sample is optionally exposed to sample processing or chemical lysis, and then the processed or lysed fluid sample is passed through a filter, where the target organism is captured. In some embodiments, sample processing is used to weaken cell walls or inactivate the sample or reduce its viscosity to facilitate processing through the filter. The filter is then washed, leaving the target organism on it. Next, the target organism is mechanically lysed (e.g., by acoustic treatment) to release nucleic acids (NA). In some embodiments, mechanical lysis includes filling the filter with glass beads to aid in the mechanical lysis of the target. The NA is then eluted from the filter, followed by nucleic acid amplification.

[0241] In workflow B, the sample is chemically lysed to obtain the NA target. In some embodiments, after chemical lysis, the NA binds to a filter in the presence of a precipitation reagent and a binding reagent. The filter is then washed with a rinsing reagent while the NA remains bound to it. Typically, the washing reagent contains a certain amount of salt, which still promotes NA binding to the filter while allowing the removal of non-target material. The filter is then eluted to remove the NA target. In some embodiments, elution is performed using a pH-neutral or alkaline buffered fluid. The target NA is then delivered to a connected reaction vessel for nucleic acid amplification.

[0242] In workflow C, the fluid sample is exposed to sample processing and / or chemical lysis of the target organism. Next, the NA released by chemical lysis binds to a filter. This step can utilize precipitation and binding reagents. Next, the filter is washed with a rinsing reagent while the NA remains bound to the filter. Typically, the washing reagent contains a certain amount of salt, which still promotes NA binding to the filter while allowing the removal of non-target material. Next, the target organisms trapped in the filter are heated and / or mechanically lysed. This can be done using acoustic treatment and can be further utilized with glass beads to promote mechanical lysis of selected target organisms. The lysed target NA is then eluted from the filter. In some embodiments, elution is performed with a pH-neutral or alkaline buffered fluid. The target NA is then delivered to a connected reaction vessel for nucleic acid amplification. Therefore, this workflow allows for the lysis of multiple different target organisms, some requiring only chemical lysis (e.g., viral targets), while others require mechanical lysis (e.g., bacteria, spores, etc.), so that all these target NAs can be released from a single sample and tested through the same sample cassette. While the above workflow describes mechanical pyrolysis following chemical pyrolysis, it should be understood that other workflows in which chemical pyrolysis occurs after mechanical pyrolysis may be utilized.

[0243] In some implementations, the sample cartridge includes an identifier with information about the appropriate workflow required for a particular assay group, causing the instrument module receiving the sample cartridge to operate according to the specified workflow.

[0244] Exemplary measurement configuration

[0245] reagents

[0246] Figure 4 An exemplary sample cartridge that can be used as part of the GENEXPERT® system is shown, in which multiple chambers are represented digitally. The exemplary cartridge may include: lyophilized reagents in the form of one or more beads as described herein; optional lysis reagents; an alkaline agent; optional binding reagents; a filtration reagent; a washing reagent; and an elution reagent. The last four reagents are named according to their function in relation to nucleic acids. Thus, for example, the binding reagent promotes the binding of nucleic acids to the substrate, and the filtration reagent promotes the filtration of nucleic acids.

[0247] In some embodiments, the lysis reagent may include a dissociating agent, a chelating agent, a buffer, an alkaline agent, or a detergent. The dissociating agent may be selected from guanidine salts (e.g., guanidine thiocyanate or guanidine hydrochloride), alkali metal perchlorates (e.g., lithium perchlorate), alkali metal iodides, magnesium chloride, urea, thiourea, formamide, or combinations thereof. The concentration of the dissociating agent may be from about 1 M to about 10 M, for example from about 2.5 M to about 7.5 M, or less than 4.5 M, less than 2 M, or less than 1 M. The chelating agent may be selected from N-acetyl-L-cysteine, ethylenediaminetetraacetic acid (EDTA), diethylenetriaminepentaacetic acid (DTPA), ethylenediamine-N,N'-disuccinic acid (EDDS), 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA), and phosphonate chelating agents. The concentration of the chelating agent may be from about 10 mM to about 100 mM and / or contain about 0.5% to about 5% of a cleavage agent. The buffer can be selected from Tris, phosphate buffer, PBS, citrate buffer, TAPS, Bicine, Tricine, TAPSO, HEPES, TES, MOPS, PIPES, cacodylate, SSC, and MES. The concentration of the buffer can be from about 5 mM to about 100 mM, for example, from about 5 mM to about 50 mM. The detergent can be selected from ionic or nonionic detergents. In some instances, the detergent contains a detergent selected from the following: N-lauroyl sarcosine, sodium dodecyl sulfate (SDS), cetyl methyl ammonium bromide (CTAB), TRITON®-X-100, n-octyl-β-D-glucopyranoside, CHAPS, n-octyl sucrose, n-octyl-β-D-maltopyranoside, n-octyl-β-D-thiopyranoside, PLURONIC® F-127, TWEEN® 20, and n-heptyl-β-D-glucopyranoside. The detergent may contain about 0.1% to about 2% of a lysis reagent, and / or about 10 mM to about 100 mM. The lysis reagent may have a pH of about 3.0 to about 5.5.

[0248] In some embodiments, the assays disclosed herein do not utilize a clinker or lysis buffer. When a clinker or lysis buffer is not used, the sample may be contacted with a buffer (or filter reagent) including, for example, saline (comprising one or more inorganic salts such as CaCl2, MgSO4, KCl, NaHCO3, NaCl, etc.), phosphate buffer, Tris buffer, 2-amino-2-hydroxymethyl-1,3-propanediol, HEPES, PBS, citrate buffer, TES, MOPS, PIPES, dimethylarsyl salt, SSC, MES, sugars or disaccharides, or combinations thereof. For example, the buffer may be a commercially available buffer, such as Hanks balanced salt solution available from Sigma Aldrich or TE buffer available from Fisher BioReagents.

[0249] In some embodiments, the alkaline agent may be selected from alkali metal hydroxides, such as sodium hydroxide or potassium hydroxide. The concentration of the alkaline agent may be from about 0.5 N to 5 N.

[0250] Binding agents can promote the binding of nucleic acids to filters, facilitating the removal of non-target materials. In some embodiments, binding agents may include binding polymers such as polyacrylic acid (PAA), polyacrylamide (PAM), polyethylene glycol (PEG), poly(sulfobetaine), or salts thereof, or combinations thereof. In some embodiments, filtering and / or washing agents may include binding agents. For example, binding agents, filtering and / or washing agents may include binding polymers (e.g., PEG 200), buffers, inorganic salts, antioxidants and / or chelating agents, defoamer SE15, sodium azide, disaccharides or disaccharide derivatives, carrier proteins, detergents, or DMSO. The conjugated polymer may be present in amounts of at least 10% v / v, at least 20% v / v, at least 30% v / v and / or less than 60% v / v, less than 40% v / v, less than 30% v / v, less than 20% v / v or less than 10% v / v of the conjugating agent, filtration agent and / or washing agent, or may fall within any range defined by any of these values, such as 10% to 60% v / v. The buffer may be selected from Tris, 2-amino-2-hydroxymethyl-1,3-propanediol, HEPES, phosphate buffer, PBS, citrate buffer, TAPS, Bicine, Tricine, TAPSO, HEPES, TES, MOPS, PIPES, dimethylarsyl salt, SSC and MES. The concentration of the buffer may be from about 5 mM to about 100 mM, for example from about 5 mM to about 50 mM. Salts, such as NaCl, KCl, or MgCl2, may be present at a concentration of about 0.05 M to about 1 M, for example, about 0.1 M to about 0.5 M. The antioxidant and / or chelating agent comprises a reagent selected from: N-acetyl-L-cysteine, ethylenediaminetetraacetic acid (EDTA), diethylenetriaminepentaacetic acid (DTPA), ethylenediamine-N,N'-disuccinic acid (EDDS), 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA), and phosphonate chelating agents. In some embodiments, the antioxidant and / or chelating agent comprises EDTA. In some embodiments, the antioxidant and / or chelating agent comprises 0.2% to about 5%, about 0.2% to about 3%, or about 0.5% to about 2%, or about 0.5% of the binding agent, filtration agent, and / or washing agent. In some embodiments, the concentration of antioxidants and / or chelating agents in the binding agent, filtering agent, or washing agent is from about 2 mM to about 50 mM or from about 5 mM to about 20 mM. In some embodiments, the detergent is an ionic detergent or a nonionic detergent. The detergent may be selected from ionic detergents or nonionic detergents.In some instances, the detergent contains a detergent selected from the following: N-lauroyl sarcosine, sodium dodecyl sulfate (SDS), hexadecylmethylammonium bromide (CTAB), TRITON®-X-100, n-octyl-β-D-glucopyranoside, CHAPS, n-octyl sucrose, n-octyl-β-D-maltopyranoside, n-octyl-β-D-thiopyranoside, PLURONIC® F-127, TWEEN® 20, Brij-35, and n-heptyl-β-D-glucopyranoside. The detergent may contain about 0.1% to about 2% of a binding agent, a filtering agent, and / or a washing agent, and / or about 10 mM to about 100 mM. The binding agent, the filtering agent, and / or the washing agent may have a pH of about 6.0 to about 8.0 (e.g., about 6.5 to about 7.5).

[0251] In some embodiments, the eluent may have a pH greater than about 9, about 10, about 11, or about 12. Using a high pH to elute nucleic acids (e.g., DNA) is unique, especially for the kit described herein, and provides improved speed and performance for the disclosed method. Speed ​​is provided by the rapid neutralization of acidic ammonium ions by a high concentration of hydroxide ions. Alkylamines have a pKa of about 10 to 11 and are immediately deprotonated at pH 12.7 to form a neutral free base on the solid surface and release cationic DNA. Another advantage of the high pH is the denaturing effect of KOH on the captured DNA or RNA. Acidic functional groups in the heterocyclic bases of DNA or RNA are immediately deprotonated and cannot form Watson-Crick bonds. Double-stranded structures and other secondary structures are disrupted but can be re-natured upon neutralization, for example, with Tris HCl. This chemical denaturation of captured genomic DNA can be advantageous for isothermal assays that do not undergo the usual thermal denaturation of PCR. The kits provided herein allow for the rapid neutralization of eluted DNA or RNA in KOH, followed by a reaction with Tris to produce a final pH of approximately 8.5 for downstream PCR or other nucleic acid assays. In some embodiments, the elution reagent may have a pH less than about 9, less than about 8.5, or less than about 8. This lower pH elution for binding DNA or RNA can be an advantage, especially for devices where rapid neutralization of KOH solutions is not feasible. RNA is known to be hydrolyzed at high pH, ​​but short exposure times to KOH can provide high-quality RNA. In some instances, the elution reagent comprises polyanionic, polycationic, ammonia, or alkali metal hydroxides. For example, the elution reagent may contain polyanionic compounds such as carrageenan, carrier nucleic acids, or combinations thereof.

[0252] In some cases, to reduce bubble formation in one or more chambers, detergent Brij can be added to one or more of the reagents described herein.

[0253] It should be understood that a variety of other reagents and initial volumes can be used for automated PCR assays of samples in the insert cassette.

[0254] Exemplary detection methods, results, and result processing

[0255] In some implementations, computer-based analysis programs are used to translate raw data generated by testing and measurement into data that has predictive value for clinicians.

[0256] The presence of PCR products can be determined by evaluating melting curve analysis using GENEXPERT® software. The melting temperature (Tm) of the curve is also considered. m The peak heights of the unwinding and dewinding processes are automatically calculated by the analysis software. If T... m Falling on the effective T specified for each target nucleic acid m If the melting curve is within the appropriate T range, it is detected as positive. m Within this range, the melting curve is considered negative. The software automatically calculates the cycle threshold (Ct), endpoint, and probe check value. The following examples provide illustrative detection limit concentrations, Ct cutoff values, and methods for determining them.

[0257] Before the PCR reaction begins, the GENEXPERT® system measures the fluorescence signal from the probe to monitor bead rehydration, reaction tube filling, probe integrity, and dye stability. If the Probe Check Control (PCC) meets the validated acceptance criteria, it passes.

[0258] In some embodiments, computer-based analysis programs are used to translate the raw data generated by the assays into data with predictive value for clinicians. Clinicians can access the predictive data using any suitable method. Therefore, in some embodiments, the invention provides the further benefit that clinicians unlikely to have received training in genetics or molecular biology do not need to understand the raw data. The data is presented directly to the clinician in its most useful form. The clinician can then immediately utilize this information to optimize care for the subject.

[0259] When using the GENEXPERT® system, results are automatically interpreted and displayed in the View Results window. Positive targets are highlighted in red, negative targets in green, and indeterminate targets in light gray. Mixed-infection samples show positive results for multiple targets. Invalid, erroneous, or no results are highlighted in light gray.

[0260] Exemplary detection methods, results, and result processing for host biomarker targets are described in U.S. Patent Publication No. 2022 / 0298572, the description of which is incorporated herein by reference.

[0261] This disclosure considers any method capable of receiving, processing, and transmitting information from the laboratory performing the assay, the information provider, the healthcare professional, and the subject. For example, in some embodiments of the invention, a sample is obtained from the subject and submitted to a testing service (e.g., a clinical laboratory at a medical institution, a genomic profiling company, etc.) located anywhere in the world (e.g., a country different from the country where the subject resides or where the information is ultimately used) to generate raw data. When the sample contains tissue or other biological material, the subject may travel to a medical center to collect the sample and send it to the testing service, or the subject may collect the sample themselves and send it directly to the testing service. When the sample includes pre-determined biological information, that information may be sent directly by the subject to the testing service (e.g., by scanning an information card containing that information via computer and transmitting the data to a computer at the profiling center using an electronic communication system). Once received by the testing service, the sample is processed and a set of test results is generated, specifically targeting the diagnostic or prognostic information desired by the subject.

[0262] Test results can be prepared in a form suitable for interpretation by the treating clinician. For example, the prepared form may not provide raw expression data, but rather represent a diagnosis or risk assessment of the subject, with or without recommendations for specific treatment options. Test results can be presented to the clinician using any suitable method. For example, in some implementations, the testing service generates a report that can be printed (e.g., at the point of care) or displayed to the clinician on a computer monitor.

[0263] In some implementations, information is first analyzed at the point of care or at a regional facility. The raw data is then sent to a central processing facility for further analysis and / or to transform the raw data into information useful to clinicians or patients. Central processing facilities offer advantages in data analysis, including privacy (all data is stored in a central facility with uniform security protocols), speed, and consistency. The central processing facility can then control the fate of the data after it has been processed. For example, using electronic communication systems, the central facility can provide the data to clinicians, subjects, or researchers.

[0264] In some implementations, subjects can directly access the data using electronic communication systems. Subjects can then choose further interventions or consultations based on the results. In some implementations, the data is used for research purposes. For example, the data can be used to further optimize the inclusion or elimination of biomarkers, which are useful indicators of specific symptoms or disease stages, or as part of a companion diagnostic process to determine treatment options.

[0265] Reagent test kit

[0266] Kits for carrying out the methods described herein are also considered. Such kits include one or more reagents that can be used to carry out any of these methods. Kits typically include packaging with one or more containers holding reagents, either as one or more separate compositions or, optionally, as a mixture where reagent compatibility permits. Kits may also include other materials that are desirable from the user's perspective, such as buffers, diluents, standards, and / or any other materials that may be used for any other steps in sample preparation, washing, or determination.

[0267] The kit preferably includes instructions for performing one or more of the screening methods described herein. The instructions included in the kit may be affixed to the packaging material or included as a packaging insert. While instructions are typically written or printed materials, they are not limited to this. Any medium capable of storing such instructions and transmitting them to the end user may be used. Such media include, but are not limited to, electronic storage media (e.g., disks, magnetic tapes, cassette tapes, chips), optical media (e.g., CD-ROMs), etc. The term "instructions" as used herein may include the address of the website providing the instructions.

[0268] In some embodiments, the kit includes primer pairs for amplifying and / or detecting selected nucleic acid targets, optionally with probes specific to those targets. Such kits may additionally include primer pairs and optional probes for detecting one or more of the aforementioned host biomarker targets. In some embodiments, these kits may include primer pairs and optional probes for detecting one or more of the aforementioned controls.

[0269] In some embodiments, the kit may include any of the above-described reagents provided with or within one or more GENEXPERT® kits. See, for example, U.S. Patents 5,958,349, 6,403,037, 6,440,725, 6,783,736, and 6,818,185 (each of which is incorporated herein by reference for the description herein). In some embodiments, reagents for measuring the target nucleic acid and reagents for detecting host biomarkers are provided in separate kits within the kit.

[0270] In some implementations, any kit described herein may include a container for samples and / or a swab for collecting samples.

[0271] Example

[0272] Example 1: Three-stage nested amplification and multi-stage detection in a single box—Method B

[0273] This embodiment illustrates one implementation scheme of method B described above: pre-amplification (stage 1). Nested amplification 1 (Stage 2) Test 1 Nested amplification 2 (stage 3) Test 2 was performed using the GENEXPERT® kit and system. As discussed above, this method is designed to detect resistance to multiple tuberculosis drugs.

[0274] Experimental Procedure

[0275] An 11-fold PCR assay was performed targeting 10 different genes in BCG and one internal control (lyophilized beads containing Bacillus globigii spores).

[0276] The assay was performed in Tris EDTA Triton (TET) buffer at concentrations of 10,000, 1,000, and 100 CFU / mL BCG to evaluate performance over a wide dynamic range of the input template.

[0277] Adding a buffer containing BCG cells to a GENEXPERT cassette containing pre-filled PCR reagents (enzymes, primers, probes) and running the assay in a GENEXPERT® instrument using predefined sample handling and PCR microfluidic steps automates the entire PCR and signal detection process.

[0278] The assay contains 16 different probes. The two probes used to detect BCG use the same fluorescent dye, resulting in a 15-color signal from PCR, which is detected in two stages as a 10+5 separation signal.

[0279] During two distinct stages of nested PCR, the real-time and post-PCR melting signals were measured twice. The final signal output (both the real-time and melting signals) was obtained as a combination of signals from the two separate nested PCR stages.

[0280] Run negative samples periodically (buffer only, no BCG) to confirm that no residual amplicon contamination has occurred.

[0281] More specifically, the following steps are performed in a box with the layout shown in Figure 2. Oligonucleotides and enzymes for pre-amplification PCR (Stage 1), PCR-1 (Stage 2), and PCR-2 (Stage 3) are provided in three separate chambers. The oligonucleotides and enzymes may be present in lyophilized beads or as a liquid PCR mixture. When using PCR beads, a liquid PCR mixture is obtained by dissolving the beads in buffer using an automated microfluidic system. The GENEXPERT box also includes a separate "master-mix chamber" (m-mix chamber), which is directly connected to the reaction tubes and is used to (i) store the PCR premix before loading it into the reaction tubes, or (ii) mix the amplicon from the previous PCR stage with the reaction mixture for subsequent PCR before loading it into the reaction tubes. Washing and elution buffers are provided in two separate chambers. The steps are as follows:

[0282] 1. Automated sample processing and DNA separation.

[0283] 2. Then push the pre-amplified PCR premix (isolated DNA + pre-amplified PCR mixture) from the pre-amplified PCR chamber into the m-mix chamber for mixing, and then aspirate the reaction chamber for the first time to perform pre-amplified PCR.

[0284] 3. Then push the pre-amplified PCR amplicons back from the reaction chamber into the pre-amplified PCR chamber (use polypropylene glass frit to securely seal it to prevent any amplicon contamination).

[0285] 4. Then, approximately 15% of the pre-amplified PCR amplicons are mixed with the PCR-1 (nested amplification 1) reaction mixture (taken from the PCR-1 chamber) in the m-mix chamber and aspirated into the reaction chamber a second time (stage 2) to perform PCR-1 (real-time / de-wiring; first stage of signal detection).

[0286] 5. Then push the PCR-1 amplicon back into the PCR-1 chamber (safely plug with polypropylene glass frit to prevent any amplicon contamination).

[0287] 6. Then wash the reaction chamber and m-mix chamber twice with TET buffer to remove any traces of amplified PCR / PCR-1 amplicons and PCR-1 premix.

[0288] 7. The PCR-2 mixture (nested amplification 2; mixture stored in the PCR-2 chamber) is now mixed with the second part of 15% pre-amplified PCR amplicon (stored in the pre-amplified PCR chamber) in the m-mix chamber and aspirated for the third time into the reaction vessel to perform PCR-2 (real-time / de-wiring; second stage of signal detection).

[0289] Figure 5The diagram shows the experimental setup for displaying biomarkers against drug resistance and the stages for detecting them. "Stage 1 primers" are pre-amplification primers. "Stage 2 analytes" and fluorophores are used for detection in PCR-1 (nested amplification / detection 1). "Stage 3 analytes" and fluorophores are used for detection in PCR-2 (nested amplification / detection 2). This experimental setup uses 16 probes to detect 11 targets.

[0290] To determine whether residual PCR amplicon contamination existed in the GENEXPERT® modules due to multiple amplicon recovery stages through the cartridge, 11 experiments were performed over 7 days using 11 different modules. 10 tuberculosis bacteria were used. 5 A colony-forming unit (CFU) run assay was performed, followed immediately by a template-free control run on the same day within one week in the same module to determine whether the initial run using a high-titer CFU resulted in any amplicon contamination in the GENEXPERT® module, which could lead to any false positive signals from the template-free control run in the same module.

[0291] result

[0292] Test sample results

[0293] Figures 6A to 6D and Figures 7A to 7D As shown, of the 15 optical signals, 13 generated both the unchaining signal and the real-time signal, while 2 generated only the expected real-time signal. These results demonstrate the effectiveness of Method B.

[0294] Residual pollution results

[0295] Table 3 below shows the results of the above residual contamination studies. Any amplicon contamination in the GENEXPERT® module during the initial run, if transferred to a new box without a template control, will result in a "false positive" signal in a negative run.

[0296] Table 3: No residual pollution

[0297]

[0298] No real-time or unwinding signals were observed in any of the template-free control runs. No signs of residual contamination were found in any of the 11 modules and 32 boxes. Therefore, the amplicon recovery microfluidic does not increase the risk of any residual amplicon contamination in the GENEXPERT® modules.

[0299] Example 2: Three-stage nested amplification and multi-stage detection in a single box—Method A (detection by HRM 2)

[0300] This embodiment illustrates the above-mentioned pre-amplification. Amplification 1 Test 1 Single amplification 2 One implementation of HRM detection 2 (high-resolution melting) is performed using the GENEXPERT® box and system. As discussed above, this method is designed to detect resistance to multiple tuberculosis drugs.

[0301] Experimental methods

[0302] In this illustrative method, three gene targets are pre-amplified together (also known as “pre-amplified PCR” or “stage 1”). Two of the targets are detected using labeled oligonucleotide probes in nested asymmetric amplification 1 (also known as “PCR-1” or “stage 2”). The third target is detected using EvaGreen HRM in nested symmetric amplification 2 (also known as “PCR-2” or “stage 3”), as shown in Table 4 below.

[0303] Table 4: Experimental Setup

[0304]

[0305] For the "positive" control reaction, use 10,000 CFU / mL BCG. For the "negative" control reaction, use TET buffer.

[0306] Three optical channels are used for target detection. Nested amplification 1 uses two channels. Nested amplification 2 uses one channel, which detects signals from the embedded dye EvaGreen.

[0307] result

[0308] The result is Figures 8A to 8B The results are shown in 9A to 9B and 10A to 10B. These results demonstrate the effectiveness of method A.

[0309] Example 3: Detection of 18 independent unwinding signals and two real-time signals in the GENEXPERT® box and system

[0310] Two octaplex / 10-color assays were performed in the same chamber to generate 18 independent melting signals and 2 real-time signals in a 10-color instrument, enabling the detection of a total of 20 independent signals in a single assay. For melting detection, three distinct amplicons were amplified from the rpoB gene in Mycobacterium tuberculosis, and one amplicon each from the fabG1, katG, and inhA promoter regions, targeted using nine melting probes. For real-time detection, the IS6110 and IS1081 genes were amplified and detected using two probes in a single channel, as they are conjugated to the same fluorescent dye. Pre-amplification PCR contained symmetrical primers targeting different genes, followed by two consecutive nested PCR stages containing the same probes and the same asymmetric primers to generate nine independent melting signals and one real-time signal in each consecutive stage, resulting in a total of 18 melting signals and 2 real-time signals measurable in a single assay. Figures 11A to 11D Clear and independent unwinding and real-time signals were obtained from nine unwinding probes and two real-time probes (detected in the same optical channel) during the two phases of the measurement.

Claims

1. A method for detecting target nucleic acid in a sample by nucleic acid amplification, the method comprising: The sample nucleic acid is contacted with a set of pre-amplification primer pairs for amplifying the target nucleic acid, wherein the target nucleic acid comprises a first set of target nucleic acids and a second set of target nucleic acids or a second set of target nucleic acids; The sample nucleic acid and pre-amplified primer pair are subjected to amplification conditions in solution to amplify any target nucleic acid present in the sample nucleic acid, thereby generating a set of double-stranded amplicon; In solution, at least a first portion of the double-stranded amplicon is contacted with a first set of nested primers and a first set of target-specific probes to form a first reaction mixture; In the first amplification, the first reaction mixture is subjected to amplification conditions to amplify any of the first group of target nucleic acids present, and a first detection, including helical analysis or real-time analysis, is performed to detect the presence of any of the first group of target nucleic acids present. In solution, at least a second portion of the double-stranded amplicon is contacted with the following: The second set of nested symmetrical primers and DNA intercalation dyes were used to amplify the second target nucleic acid. or Each of the following is used to amplify the second set of target nucleic acids: a second set of nested symmetrical or asymmetrical primers, and a second set of target-specific real-time or unwinding probes. To form a second reaction mixture; In the second amplification, the second reaction mixture is subjected to amplification conditions to amplify it in the presence of the second target nucleic acid or the second group of target nucleic acids, and a second detection is performed, the second detection comprising: High-resolution melting analysis to detect the second target nucleic acid in its presence; or Unwinding analysis or real-time analysis to detect the presence of any of the second group of target nucleic acids; The first amplification and first detection, as well as the second amplification and second detection, are performed sequentially, but can be performed in any order.

2. A kit for detecting target nucleic acids in a sample, the kit comprising: A housing containing multiple chambers, wherein the multiple chambers include: A sample chamber having at least one fluid outlet in fluid communication with another of the plurality of chambers; An optional lysis chamber, which is in fluid communication with the sample chamber, wherein the sample chamber and the lysis chamber are the same; The pre-amplification reagent chamber contains a first set of pre-amplification primer pairs for amplifying target nucleic acids, wherein the target nucleic acids comprise a first set of target nucleic acids and a second set of target nucleic acids. The first reagent chamber contains a first set of nested primers and a first set of target-specific probes for performing the first amplification; and The second reagent chamber contains a second set of nested primers, wherein: The second set of nested primers contains symmetrical primers for amplifying the second target nucleic acid, and the second reagent chamber also contains a DNA intercalation dye; or The second set of nested primers contains primers for symmetrical or asymmetric amplification of the second set of target nucleic acids and a second set of target-specific probes; A reaction vessel, fluidly connected to multiple chambers of the housing, and configured for: i) amplification of nucleic acids and ii) detection and identification of one or more amplified products by melt-blown assay and / or real-time PCR, wherein the housing is configured for multi-stage detection; and A filter is provided in the fluid path between the pyrolysis chamber and the reaction vessel, or in the fluid path between the sample chamber and the reaction vessel.

3. A box-based method for detecting target nucleic acids in a sample within a box according to claim 2, the method comprising: The sample is placed in the sample chamber of the box; If the sample contains cells, the cells in the sample are lysed with one or more lysis reagents present in at least one of the plurality of chambers, or the cells are captured in a filter within the box and lysed by sonication to release the sample nucleic acids; If the sample contains cell-free nucleic acid, the free nucleic acid is captured in a nucleic acid capture chamber and the captured nucleic acid is eluted after washing to remove impurities; The sample nucleic acid is brought into contact with the pre-amplified primer pair within the reaction vessel; The sample nucleic acid and pre-amplified primer pair are subjected to amplification conditions in solution to amplify any target nucleic acid present in the sample nucleic acid, thereby generating a set of double-stranded amplicon; The double-stranded amplicon is allowed to flow into the first chamber of the plurality of chambers; A first portion of the double-stranded amplicon is extracted from the first chamber of the plurality of chambers and contacted in solution with a first set of nested primers and a first set of target-specific probes to form a first reaction mixture within the reaction vessel; In the first amplification, the first reaction mixture is subjected to amplification conditions to amplify any of the first group of target nucleic acids present, and a first detection, including helical analysis or real-time analysis, is performed to detect the presence of any of the first group of target nucleic acids present. The first reaction mixture is allowed to flow into at least one of the plurality of chambers, and the reaction vessel is then rinsed. The second portion of the double-stranded amplicon is extracted from the first chamber of the plurality of chambers, and the second portion of the double-stranded amplicon is contacted in solution with the following: The second set of nested symmetrical primers and DNA intercalation dyes were used to amplify the second target nucleic acid. or The second set of nested symmetrical or asymmetrical primers and the second set of target-specific probes are used to amplify the second set of target nucleic acids. To form a second reaction mixture within the reaction vessel; In the second amplification, the second reaction mixture is subjected to amplification conditions to amplify it in the presence of the second target nucleic acid or the second group of target nucleic acids, and a second detection is performed, the second detection comprising: High-resolution melting analysis to detect the second target nucleic acid in its presence; or Unwinding analysis or real-time analysis to detect the presence of any of the second group of target nucleic acids; The first amplification and first detection, as well as the second amplification and second detection, are performed sequentially, but can be performed in any order.

4. The method of claim 1, the cassette of claim 2, or the cassette-based method of claim 3, wherein the second set of nested primers comprises symmetric primers for amplifying the second target nucleic acid, accompanied by the DNA intercalation dye, and the high-resolution melting analysis is performed, or the cassette is configured to perform high-resolution melting analysis to detect the second target nucleic acid in its presence.

5. The method of claim 1, the box of claim 2, or the box-based method of claim 3, wherein the second set of nested primers comprises asymmetric primers for amplifying the second set of target nucleic acids and is accompanied by the second set of target-specific probes, the second set of target-specific probes being unwinding detection probes and performing unwinding analysis, or the box is configured to perform unwinding analysis to detect the presence of any of the second set of target nucleic acids present.

6. The method of claim 1, the box of claim 2, or the box-based method of claim 3, wherein the second set of nested primers comprises symmetric primers for amplifying the second set of target nucleic acids and is accompanied by the second set of target-specific probes, the second set of target-specific probes being real-time probes and being analyzed in real time, or the box is configured to perform real-time analysis to detect the presence of any of the second set of target nucleic acids present.

7. The method, cassette, or cassette-based method of any one of claims 5 to 6, wherein the method detects 10 to 20 target nucleic acids in each of the first reaction mixture or the second reaction mixture, or the cassette is configured to detect 10 to 20 target nucleic acids in each of the first reaction mixture or the second reaction mixture.

8. The method, cassette, or cassette-based method of claim 7, wherein the method detects 18 to 20 target nucleic acids in each of the first reaction mixture or the second reaction mixture, or the cassette is configured to detect 18 to 20 target nucleic acids in each of the first reaction mixture or the second reaction mixture.

9. The method, cassette, or cassette-based method of claim 8, wherein the method detects 36 to 40 target nucleic acids in a single cassette, or the cassette is configured to detect 36 to 40 target nucleic acids in a single cassette.

10. The box of any one of claims 7 to 9, wherein the reaction container comprises a reaction chamber, and the detection of the target nucleic acid is performed in the reaction chamber.

11. The kit of any one of claims 7 to 9, wherein the reaction container comprises up to four reaction chambers, and the detection of the target nucleic acid is performed in the up to four reaction chambers.

12. The method, cassette, or cassette-based method of any one of claims 5 to 7, wherein the pre-amplification primers comprise primers specific to one or more of the following drug resistance genes: amikacin resistance gene rrs; aminoglycoside resistance genes rrs and eis; bedaquiline resistance genes atpE and Rv0678; fluoroquinolone resistance genes gyrA and gyrB; capreomycin resistance genes gidB, rrs, and tlyA; clofazimine resistance gene Rv0678; delamani resistance genes fbiA and ddn; ethionamide resistance gene inhA promoter and ethA; ethambutol resistance gene embB; isoniazid resistance genes fabG1, inhA promoter, and katG; linezolid resistance genes rplC and rrl; pyrazinamide resistance gene pncA; rifampin resistance gene rpoB; and streptomycin resistance genes gidB, rrs, and rpsL.

13. The method, cassette, or cassette-based method of any one of claims 5 to 7, wherein the pre-amplification primers comprise primers specific to one or more of the following: the rpoB RRDR gene, rpoB 491 gene, rpoB V170 gene, IS6110 gene, IS1081 gene, fabG1 gene, inhA promoter, katG gene, gyrA gene, gyrB gene, pncA gene, rplC gene, rrl gene, atpE gene, and Rv0678 gene of Mycobacterium tuberculosis.

14. The method, cassette, or cassette-based method of claim 13, wherein the pre-amplification primers comprise primers specific to each of the following: Mycobacterium tuberculosis rpoB RRDR gene, rpoB 491 gene, rpoB V170 gene, IS6110 gene, IS1081 gene, fabG1 gene, inhA promoter, katG gene, gyrA gene, gyrB gene, pncA gene, rplC gene, rrl gene, atpE gene, and Rv0678 gene.

15. The method, cassette, or cassette-based method of claim 13 or 14, wherein the first set of nested primers and the first set of target-specific probes comprise primers and probes specific to one or more of the following: rpoB RRDR, rpoB 491, and rpoB V170 targets of the rpoB gene, the IS6110 gene, the IS1081 gene, the fabG1 gene, the inhA promoter, the katG gene, the gyrA gene, the gyrB gene, the Rv0678 gene, and the pncA gene.

16. The method, cassette, or cassette-based method of claim 14, wherein the first set of nested primers and the first set of target-specific probes comprise primers and probes specific to each of the following: rpoB RRDR, rpoB 491, and rpoB V170 targets of the rpoB gene, the IS6110 gene, the IS1081 gene, the fabG1 gene, the inhA promoter, the katG gene, the gyrA gene, the gyrB gene, the Rv0678 gene, and the pncA gene.

17. The method, cassette, or cassette-based method of any one of claims 13 to 16, wherein the second set of nested symmetrical primers is specific for the Rv0678 gene or pncA gene of Mycobacterium tuberculosis.

18. The method, cassette, or cassette-based method of claim 13 or 14, wherein the first set of nested primers and the first set of target-specific probes comprise primers and probes specific to one or more of the following: rpoB RRDR and rpoB V170 targets of the rpoB gene of Mycobacterium tuberculosis, the IS6110 gene, the IS1081 gene, the inhA promoter, and the katG gene.

19. The method, cassette, or cassette-based method of claim 18, wherein the first set of nested primers and the first set of target-specific probes comprise primers and probes specific to each of the following: rpoB RRDR and rpoB V170 targets of the rpoB gene of Mycobacterium tuberculosis, the IS6110 gene, the IS1081 gene, the inhA promoter, and the katG gene.

20. The method, cassette, or cassette-based method of any one of claims 13 to 16, 18, and 19, wherein the second set of nested primers and the second set of target-specific probes comprise primers and probes specific to one or more of the following: the rpoB 491 target of the rpoB gene of Mycobacterium tuberculosis, the fabG1 gene, the gyrA gene, and the gyrB gene.

21. The method, cassette, or cassette-based method of claim 20, wherein the second set of nested primers and the second set of target-specific probes comprise primers and probes specific to each of the following: the rpoB 491 target of the rpoB gene of Mycobacterium tuberculosis, the fabG1 gene, the gyrA gene, and the gyrB gene.

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