WO2025179142A1 - Maximizing optical channel capabilities for target detection - Google Patents

Abstract

Compositions, methods, and systems for detecting melt temperature signatures for at least two target regions of nucleic acid in a multiplex amplification reaction are provided. The methods include contacting the nucleic acid with sets of primers and sets of two or more probes for detecting each target region, wherein each of the two or more probes in a set comprises a detectable label that emits light at the same wavelength; subjecting the nucleic acid, primers, and probes to amplification conditions to amplify the target regions; and generating and detecting a melt temperature signature specific for each target region present, wherein the melt temperature signature comprises a plurality of melt profiles generated from the set of two or more probes hybridizing to the amplified target region.

Description

MAXIMIZING OPTICAL CHANNEL CAPABILITIES FOR TARGET DETECTION

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application is being filed on February 21, 2025, as a PCT International application and claims the benefit of and priority to U.S. Provisional Application No. 63/556,730, filed February 22, 2024; the disclosure of which is hereby incorporated by reference in its entirety.

FIELD

[0002] The present invention relates generally to the area of detecting one or more target regions of nucleic acid by polymerase chain reaction, and more particularly to methods and systems for generating and detecting multiple unique melt temperature signatures for target region of nucleic acid in a multiplex amplification reaction.

BACKGROUND

[0003] Molecular methods can provide higher sensitivity and faster time to results than culture methods. However, current nucleic acid amplification methods have limitations because amplification reaction and signal detection require a controlled environment and precise measurement with expensive instruments. Thus, the methods are often cost-prohibitive for use in point-of-care situations. Additionally, some methods are not optimized for detection of multiplexed target regions in single patient samples. Detection of multiplexed targets may be accomplished by signal multiplexing in single-pot reactions (fluorescent spectral multiplexing, arrays of electrochemical detectors), physical separation of multiple reactions into unique reaction vessels, or a combination thereof. Physical separation of multiple reactions into unique reaction vessels can lead to erroneous results due to differences in sampling or in assay conditions during reactions, which can confound efforts to make differential diagnoses. Furthermore, in the case of a CLIA-waived test, no more than three simple steps must be required by the user to simultaneously query a panel of target regions using a single patient sample. Physical separation of samples into discrete chambers quickly becomes infeasible for CLIA-waived tests, unless a complicated device or disposable automatically handles processing.

[0004] Multiplexing can overcome some of these difficulties, but presents its own technical challenges, particularly with attempts to assay for more than a few pathogens in a single reaction mixture. For example, multiplexes are often limited by the choice of available fluorophores for target detection. Since the early 2000s, however. DNA-detection technologies have bifurcated into either massively multiplexed but slow systems (next-generation sequencing (NGS) and microarrays), or rapid assays with limited capacity for multiplexing (quantitative PCR (qPCR) and isothermal amplification). Spectral multiplexing with fluorescence can reduce the number of unique reactions required to detect a panel of target regions, but spectral multiplexing LAMP reactions has required dramatic sacrifices in assay speed or signal strength, dampening prospects for successful application to point-of-care testing.

[0005] The methods, compositions, and devices presented herein achieve rapid, sensitive, qualitative, and optionally quantitative detection of many target regions (DNA and RNA) from a single sample, in some embodiments, using a closed and affordable instrument.

SUMMARY

[0006] Multiplexing is often limited by the choice of available fluorophores for target detection. This disclosure offers a creative way of maximizing the detection of a wide number of targets by getting unique double, triple or more melt profile signatures from probes with the same fluorophore. This multi-signature approach is based on a single fluorophore and can be used alone or in conjunction with unique T_m signatures from other probes with different fluorophores in a multiplex PCR reaction to uniquely identify many nucleic acid sequences (pathogens), enabling high-level multiplexing. Getting unique melt signatures using two or more probes (e.g., sloppy molecular beacons) with the same fluorophore and quencher options in a multiplex will extend the functionality of fluorophore-based differentiation and identification of targets using melt curve analysis. This can be very beneficial when limited by the number of fluorophores available for target detection and can be applied to large multiplex panels such as tuberculosis panels, nontuberculosis panels, gastric panels, sepsis pathogen panels, respiratory panels, intestinal pathogen panels, immunology panels, metabolic syndrome panels, nephrotoxicity analysis panels, other toxicity panels, alzheimer’s disease panels, and such the like.

[0007] Various embodiments contemplated herein may include, but need not be limited to, one or more of the following:

[0008] Particularly, the present disclsoure describes methods for detecting melt temperature signatures for at least two target regions of nucleic acid in a multiplex amplification reaction, the method comprising: contacting the nucleic acid with sets of primers and sets of two or more probes for detecting each target region, wherein each of the two or more probes in a set comprises a detectable label that emits light at the same wavelength; subjecting the nucleic acid, primers, and probes to amplification conditions to amplify the target regions; and generating and detecting a melt temperature signature specific for each target region present, wherein the melt temperature signature comprises a plurality of melt profiles generated from the set of two or more probes hybridizing to the amplified target region. In certain embodiments, the method comprises contacting the nucleic acid with sets of primer and sets of two or more probes for simultaneously detecting the at least 2 target regions, preferably at least 5 target regions, more preferably at least 10 target regions, wherein the detectable label used in the sets of two or more probes are the same. Multiple probes with a same fluorophore multiplexed with other probe with different fluorophore in the multiplex will generate even more unique melt I signatures. Accordingly, in certain embodiments, the method comprises contacting the nucleic acid with sets of primer and sets of two or more probes for simultaneously detecting the at least 2 target regions, preferably at least 5 target regions, more preferably at least 10 target regions, wherein the detectable label used in the sets of two or more probes are different. In other certain embodiments, the method comprises contacting the nucleic acid with sets of primer and sets of two or more probes for simultaneously detecting at least 5 target regions, more preferably at least 10 target regions, wherein the detectable label used in a plurality of the sets of two or more probes are the same, and the detectable label used in a plurality of the sets of two or more probes are different.

[0009] The two or more probes in a set can be selected from overlapping (staggered) hybridizing probes, sequential hybridizing probes, spaced out hybridizing probes, or a combination thereof. When combined with other probes with different fluorophore(s) can maximize the ability to identify several sequences uniquely. The inventors have shown that the melt peaks of the two or more overlapping (staggered), sequential, or spaced out probes within the same fluorescent channel in a multiplex reaction system gave the same T_m as single probes, thus giving broader coverage of specific targets/variants without using additional channels. Each target region that hybridizes to the two or more probes in a set is at least forty (40) nucleotides or at least fifty (50) nucleotides in length. The sets of two or more probes can comprise a molecular beacon probe, a linear probe, a FRET (TaqMan) probe, or a combination thereof. In some instances, the sets of two or more probes comprise at least two sloppy molecular beacon probes, at least two linear probes, or a sloppy molecular beacon probe and a linear probe. The detectable label comprises a fluorescent dye and a quencher molecule. The melt temperature signature for each target region comprises at least one melt peak temperature (T_m), preferably at least two T_ms. In some instances, when the melt temperature signature comprises two or more T_ms. the T_ms are separated by at least 4°C.

[0010] In addition to detecting the target regions, the methods can further comprise differentiating and identifying each target region. The target region can be selected from a pathogenic nucleic acid such as bacterial nucleic acid, viral nucleic acid, fungal nucleic acid, or a combination thereof. In some embodiments, the target regions include a Mycobacterium tuberculosis gene, a nontuberculous Mycobacterium gene, or a combination thereof. In some examples, the target regions include a nontuberculous Mycobacterium gene selected from Mycobacterium abscessus. Mycobacterium avium, Mycobacterium chelonae. Mycobacterium fortuitum. Mycobacterium gordonae, Mycobacterium intracellulare, Mycobacterium kansasii, Mycobacterium simiae, Mycobacterium xenopi, or a combination thereof.

[0011] The sample can be selected from a sputum sample, a nasal aspirate sample, a nasal wash sample, a nasal swab sample, a nasopharyngeal swab sample, a saliva sample, an oropharyngeal swab sample, a throat swab sample, a bronchoalveolar lavage sample, a bronchial aspirate sample, a bronchial wash sample, an endotracheal aspirate sample, an endotracheal wash sample, a tracheal aspirate sample, a nasal secretion sample, a mucus sample, a pleural effusion sample, a cerebrospinal fluid sample, a stool sample, a tissue biopsy sample, a breath sample, or a combination thereof.

[0012] Amplification can comprise non-isothermal amplification, optionally by thermal cycling or temperature oscillation. In some examples, method is performed via real-time PCR. In some embodiments, the method is a point-of-care method and can be performed within 150 minutes, within 140 minutes, within 130 minutes, or within 120 minutes of collecting the sample from the subject.

[0013] The methods described herein can be automated or semi-automated. For example, the methods can be a cartridge-based method and comprises placing the nucleic acid sample in a sample chamber of a cartridge; and if the sample comprises cells, lysing the cells in the sample with one or more lysis reagents present within at least one of the plurality of chambers or capturing the cells in a filter within the cartridge and lysing the cells by means of ultrasonication to release sample nucleic acids; or if the sample comprises cell -free nucleic acid, capturing the free nucleic acids in a nucleic acid capture chamber and eluting the captured nucleic acid after washing to remove impurities.

[0014] Cartridges for detecting melt temperature signatures for at least two target regions of nucleic acid in a multiplex amplification reaction according to a methods disclosed are also provided. The cartridges can comprise a cartridge body comprising a plurality of chambers therein, wherein the plurality of chambers includes: a sample chamber having at least a fluid outlet in fluid communication with another chamber of the plurality; and an optional lysis chamber in fluidic communication with the sample chamber, optionally wherein the sample chamber and lysis chamber are the same; a reaction vessel fluidically coupled to the plurality of chambers of the cartridge body and configured for: i) amplification of nucleic acid and ii) detection and identification of a plurality of amplification products via real-time PCR; a filter disposed in a fluidic path between the lysis chamber, if present, or the sample chamber, and the reaction vessel, and sets of primers and sets of two or more probes for detecting each target region, wherein each of the two or more probes in a set comprises a detectable label that emits light at the same wavelength. The cartridge can be a Clinical Laboratory' Improvement Amendments (CLIA)-compliant cartridge. The cartridge can be configured to carry out non-isothermal amplification, optionally by thermal cycling or temperature oscillation, preferably real-time PCR amplification.

[0015] The lysis chamber can comprise one or more lysis reagents for releasing nucleic acid. The reaction vessel can comprise one or more reaction chambers for amplification and detection of the amplification products. In some embodiments, the reaction vessel comprises one reaction chamber for amplification and detection of the amplification products. According, in some instances, the nucleic acid, primers, and probes can be present in a single reaction solution, and wherein generating and detecting the melt temperature signatures for the target regions are from the single reaction solution. The reaction chamber can be configured to detect a single amplification product. In some embodiments, each reaction chamber is configured to detect a plurality of amplification products.

[0016] Kits for detecting melt temperature signatures for at least two target regions of nucleic acid in a multiplex amplification reaction according to the methods herein are also provided. The kits can comprise sets of primers and sets of two or more probes for detecting each target region, wherein each of the two or more probes in the set compnses a detectable label that emits light at the same wavelength.

BRIEF DESCRIPTION OF THE DRAWINGS

[0017] FIG. 1 is a schematic diagram showing an example staggered probe approach to get unique melt temperature (T_m) values for a particular site of interest. [0018] FIGS. 2A-2C are graphs showing results from wet lab experimental data with staggered probes in a single optical channel. As seen from the GeneXpert curves, two distinct T_m values were reported for AT abscessus (FIG. 2A), M. asiaticum (FIG. 2B), and AT gastri (FIG. 2C).

[0019] FIGS. 3A-3C show an overview of a sample cartridge with a valve assembly configured for performing differing sample processes, including chemical lysing of targets, which is configured for PCR and integrated nucleic acid analysis in accordance with some embodiments of the invention. FIG. 3A shows the sample cartridge body with reaction vessel, FIG. 3B shows an exploded view of the sample cartridge, and FIG. 3C shows components of the valve assembly, in accordance with some embodiments.

DETAILED DESCRIPTION

[0020] The present disclosure describes methods, compositions, devices, and systems that facilitate detection of melt temperature signatures for at least two target regions of nucleic acid in a multiplex amplification reaction. The methods, compositions, devices, and systems are readily automated and can be employed in point-of-care devices, enabling detection and discrimination between target nuclei acid in the multiplex reaction, so that the appropriate treatment can be administered in a timely manner. The approach relies on nucleic acid amplification to detect the target regions, which entails contacting each target region with a set of primer and a set of two or more probes, wherein each of the two or more probes in the set for a target region comprises a detectable label that emits light at the same wavelength. The method further comprises subjecting the nucleic acid, primers, and probes to amplification conditions to amplify the target regions; and generating and detecting a melt temperature signature specific for each target region, wherein the melt temperature signature comprises a plurality of melt profiles generated from the set of two or more probes hybridizing to the amplified target region.

Definitions

[0021] Terms used in the claims and specification are defined as set forth below unless otherwise specified.

[0022] The term '‘nucleic acid’’ refers to a nucleotide polymer, and unless otherwise limited, includes analogs of natural nucleotides that can function in a similar manner (e.g.. hybridize) to naturally occurring nucleotides.

[0023] The term nucleic acid also includes any form of DNA or RNA, including, for example, genomic DNA; complementary DNA (cDNA), which is a DNA representation of mRNA, usually obtained by reverse transcription of messenger RNA (mRNA) or by amplification; DNA molecules produced synthetically or by amplification; mRNA; and non-coding RNA.

[0024] The term nucleic acid further encompasses double- or triple-stranded nucleic acid complexes, as well as single-stranded molecules. In double- or triplestranded nucleic acid complexes, the nucleic acid strands need not be coextensive (i.e., a double-stranded nucleic acid need not be double-stranded along the entire length of both strands).

[0025] The term nucleic acid also encompasses any modifications thereof, such as by methylation and/or by capping. Nucleic acid modifications can include addition of chemical groups that incorporate additional charge, polarizability, hydrogen bonding, electrostatic interaction, and functionality to the individual nucleic acid bases or to the nucleic acid as a whole. Such modifications may include base modifications such as 2’-position sugar modifications, 5-position pyrimidine modifications, 8-position purine modifications, modifications at cytosine exocyclic amines, substitutions of 5- bromo-uracil, sugar-phosphate backbone modifications, unusual base pairing combinations such as the isobases isocytidine and isoguanidine, and the like.

[0026] More particularly, in some embodiments, nucleic acids, can include poly deoxyribonucleotides (containing 2-deoxy-D-ribose), polyribonucleotides (containing D-ribose), and any other type of nucleic acid that is an N- or C-glycoside of a purine or pyrimidine base, as well as other polymers containing nonnucleotidic backbones, for example, polyamide (e.g., peptide nucleic acids (PNAs)) and polymorpholino polymers (see, e.g.. Summerton and Weller (1997) "Morpholino Antisense 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: 171-172). and other synthetic sequence-specific nucleic acid polymers providing that the polymers contain nucleobases in a configuration which allows for base pairing and base stacking, such as is found in DNA and RNA. The term nucleic acid also encompasses locked nucleic acids (LNAs), which are described in U.S. Patent Nos. 6,794,499, 6,670,461, 6,262,490, and 6,770,748, which are incorporated herein by reference in their entirety for their disclosure of LNAs.

[0027] The nucleic acid(s) can be derived from a completely chemical synthesis process, such as a solid phase-mediated chemical synthesis, from a biological source, such as through isolation from any species that produces nucleic acid, or from processes that involve the manipulation of nucleic acids by molecular biology tools, such as DNA replication, PCR amplification, reverse transcription, or from a combination of those processes.

[0028] As used herein, the term “gene” encompasses coding sequences, introns, and any associated control sequences that participate in the expression of the coding sequences.

[0029] As used herein, the term “complementary” refers to the capacity for precise pairing between two nucleotides; i.e., if a nucleotide at a given position of a nucleic acid is capable of hydrogen bonding with a nucleotide of another nucleic acid to form a canonical base pair, then the two nucleic acids are considered to be complementary to one another at that position. Complementarity between two singlestranded nucleic acid molecules may be “partial,” in which only some of the nucleotides bind, or it may be complete when total complementarity exists betw een the two single-stranded molecules. The degree of complementarity between nucleic acid strands has significant effects on the efficiency and strength of hybridization between nucleic acid strands.

[0030] “Selective hybridization” or “selective annealing” refers to the binding of a nucleic acid to a target region in the absence of substantial binding to other nucleic acids present in the hybridization mixture under defined stringency conditions. Those of skill in the art recognize that relaxing the stringency of the hybridization conditions allows sequence mismatches to be tolerated.

[0031] In some embodiments, hybridizations are carried out under stringent hybridization conditions. The phrase “stringent hybridization conditions” generally refers to a temperature in a range from about 5°C to about 20°C or 25°C below than the melting temperature (T_m) for a specific sequence at a defined ionic strength and pH. As used herein, the T_m is the temperature at which a population of double-stranded nucleic acid molecules becomes half-dissociated into single strands. Methods for calculating the Tm of nucleic acids are well known in the art (see, e.g., 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, 2^nd Ed., Vols. 1-3, Cold Spring Harbor Laboratory), both incorporated herein by reference for their descriptions of stringent hybridization conditions. As indicated by standard references, a simple estimate of the T_m value may be calculated by the equation: T_m = 81.5 + 0.41 (% G+C), when a nucleic acid is in aqueous solution at 1 M NaCl (see, e.g., Anderson and Young, Quantitative Filter Hybridization in Nucleic Acid Hybridization (1985)). The melting temperature of a hybrid, and thus the conditions for stringent hybridization, is affected by various factors such as the length and nature (e.g., DNA, RNA, base composition) of the primer or probe and nature of the target region (e.g., DNA, RNA. base composition, present in solution or immobilized, and the like), as well as the concentration, or presence or absence of salts and other components (e.g., formamide, dextran sulfate, polyethylene glycol, and the like). The effects of these factors are well known and are discussed in standard references in the art. Illustrative stringent conditions suitable for achieving specific hybridization of most sequences may include a temperature of at least about 60°C and a salt concentration of about 0.2 molar at pH of about 7. T_m calculation for oligonucleotide sequences based on nearest-neighbors thermodynamics can be carried out 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 (which is incorporated by reference herein for this description).

[0032] The term “oligonucleotide” is used to refer to a nucleic acid that is relatively short, generally shorter than 200 nucleotides, more particularly, shorter than 100 nucleotides, most particularly, shorter than 50 nucleotides, and any value in between (e.g., about 58 nucleotides, or about 24 nucleotides). Typically, oligonucleotides are single-stranded DNA molecules.

[0033] The term “primer” refers to an oligonucleotide that is capable of hybridizing (also termed “annealing”) with a nucleic acid and serving as an initiation site for nucleotide (RNA or DNA) polymerization under appropriate conditions (i.e.. in the presence of four different nucleoside tnphosphates and an agent for polymerization, such as DNA or RNA polymerase or reverse transcriptase) in an appropriate buffer and at a suitable temperature. The appropriate length of a primer depends on the intended use of the primer, but primers are typically at least 7 nucleotides long and, in some embodiments, range from about 10 to about 30 nucleotides, or, in some embodiments, from about 10 to about 60 nucleotides in length. In some embodiments, primers can be, e.g., about 15 to about 50 nucleotides long. Short primer molecules generally require cooler temperatures to form sufficiently stable hybrid complexes with the template. A primer need not reflect the exact sequence of the template but must be sufficiently complementary to hybridize with a template.

[0034] A primer is said to “anneal to” or “hybridize to” another nucleic acid if the primer, or a portion thereof, hybridizes to a nucleotide sequence within the nucleic acid. The statement that a primer hybridizes to a particular nucleotide sequence is not intended to imply that the primer hybridizes either completely or exclusively to that nucleotide sequence. For example, in some embodiments, amplification primers used herein are said to “anneal to” or be “specific for” a nucleotide sequence.” This description encompasses primers that anneal wholly to the nucleotide sequence, as well as primers that anneal partially to the nucleotide sequence.

[0035] The term “primer pair” refers to a set of primers including a 5’ “upstream primer” or “forward primer” that hybridizes with the complement of the 5’ end of the DNA sequence to be amplified and a 3 ’ “downstream primer” or “reverse primer” that hybridizes with the 3’ end of the sequence to be amplified. As will be recognized by those of skill in the art. the terms “upstream” and “downstream” or “forward” and “reverse” are not intended to be limiting, but rather provide illustrative orientations in some embodiments.

[0036] A “probe” is a nucleic acid capable of binding to a target region of complementary sequence through one or more types of chemical bonds, generally through complementary base pairing, usually through hydrogen bond formation, thus forming a duplex structure. The probe can be labeled with a detectable moiety to permit facile detection of the probe, particularly once the probe has hybridized to its complementary target. Alternatively, however, the probe may be unlabeled, but may be detectable by specific binding with a ligand that is labeled, either directly or indirectly. Probes can vary significantly in size.

[0037] "Molecular beacon probe,” as used herein refers to a hybridization probe that forms a stem and loop structure. The molecular beacon probe may range in length from about 5 nucleotides to about 1000 nucleotides, most preferably from about 10 to about 50 nucleotides in length The molecular beacon probe has a 5' arm, a loop portion that is a probe sequence, and a 3' arm. The 5' and 3' arms are complementary to each other but not to the loop portion or the target and bind to each other to form the stem of the molecular beacon probe. The arms are preferably from about 3-10 nucleotides in length, and more preferably from about 5-7 nucleotides in length. Probes may bind target sequences lacking complete complementarity with the probe sequence depending upon the stnngency of the hybridization conditions. The molecular beacon probes of the present invention are mismatch tolerant and may contain a substantial number of mismatched base pairs relative to the target, and thus are also referred to herein as “sloppy molecular beacons” or “SMBs”. In a preferred embodiment, a fluorophore is attached to one end of the molecular beacon probe and a non-fluorescent quencher moiety is attached to the other end. For example, the molecular beacon probe may have a fluorophore attached to the 5'-end and a quencher moiety attached to the 3'-end, or a fluorophore attached to the 3'-end and a quencher moiety attached to the 5'-end.

Fluorophores are known in the art and include fluorescein, rhodamine and cyanine derivatives. Quenchers are known in the art and include Black Hole Quenchers (Biosearch Technologies, Novato, Calif).

[0038] As used herein with reference to a portion of a primer or a nucleotide sequence within the primer, the term “specific for” a nucleic acid, refers to a primer or nucleotide sequence that can specifically anneal to the target region under suitable annealing conditions.

[0039] The term “target” is used herein with reference to “target regions,” as well as “target organisms.” The former refers to nucleic acids to be detected, and the latter refers to organisms to be detected. The term, “target region” is generally used herein to refer to a segment of nucleic acid that is defined by a primer pair and that gives rise to an amplicon produced in an amplification reaction; the term “amplification target” is also used herein to refer to this type of target region. Primers and probes are also said to “target” nucleic acid sequences, and so these sequences can also be understood as “target regions.” Additionally, primers and probes are said to “target” or “be specific for” genes. In this usage, the primers and probes can be used to detect the presence of a particular gene by specifically hybridizing to a portion of the gene that indicates its presence. The meaning of “target” and “target regions” will be clear to one of skill in the art from the context in which the term is employed. In some embodiments, multiple target regions can be detected to detect a single target organism. In some embodiments, a single target region can be detected to detect a single target organism. In some embodiments, an assay can employ multiple target regions for one or more target organisms and single target regions for one or more different target organisms.

[0040] Amplification according to the present teachings encompasses any means by which at least a part of at least one target region is reproduced, typically in a template-dependent manner, including without limitation, a broad range of techniques for amplifying nucleic acid sequences, either linearly or exponentially. Illustrative means for performing an amplifying step include PCR, nucleic acid strand-based amplification (NASBA). two-step multiplexed amplifications, rolling circle amplification (RCA), and the like, including multiplex versions and combinations thereof, for example 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 (HD A), and the like. Descriptions of such techniques can be found in, among 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, N.J. (2002); Abramson, et al., Curr Opin Biotechnol.. 1993 Feb.;4(l):41-7. U.S. Pat. No. 6,027,998; U.S. Pat. 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., Science 252: 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)1 Belgrader, Barany, and Lubin, Development of a Multiplex Ligation Detection Reaction DNA Typing Assay, Sixth International Symposium on Human Identification, 1995 (available on the world wide web at: promega.com/geneticidproc/ussymp6proc/blegrad.html- ); LCR Kit Instruction Manual, Cat. #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.. Proc Natl 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. 2003 Feb.;13(2):294-307, and Landegren, et al., Science 241 : 1077-80 (1988). Demidov, V., Expert Rev Mol Diagn. 2002 Nov.;2(6):542-8, Cook, et al., J Microbiol Methods. 2003 May;53(2): 165-74, Schweitzer, et al., Curr Opin Biotechnol. 2001 Feb.;12(l):21-7, U.S. Pat. No. 5,830,711, U.S. Pat. No. 6.027,889, U.S. Pat. No. 5,686,243, PCT Publication No. W02000/56927A3, and PCT Publication No.

WO 1998/03673 Al.

[0041] In some embodiments, amplification comprises at least one cycle of the sequential procedures of: annealing at least one primer with complementary or substantially complementary sequences in at least one target region; synthesizing at least one strand of nucleotides in a template-dependent manner using a polymerase; and denaturing the newly-formed nucleic acid duplex to separate the strands. The cycle may or may not be repeated. Amplification can comprise thermocycling or can be performed isothermally.

[0042] As used herein, the term “amplification conditions” refers to conditions that promote amplification of a target region in the presence of suitable primers.

[0043] As used herein, “in solution” means not immobilized on a substrate of any kind, for example, a bead or a surface in a cassette, such as a chamber wall.

[0044] A “multiplex amplification reaction” is one in which two or more nucleic acids distinguishable by sequence are amplified simultaneously.

[0045] The term “qPCR” is used herein to refer to quantitative real-time polymerase chain reaction (PCR), which is also known as “real-time PCR” or “kinetic polymerase chain reaction;” all terms refer to PCR with real-time signal detection. [0046] The term “melt profile'’ as used herein to refers to the morphological characteristics of the melt curve, particularly, a geometry change (e.g., melt rate) throughout the curve that describes the dissociation characteristics of a segment of double-stranded nucleic during heating. A melt profile may show one or more melt phases or a melt temperature (T_m).

[0047] The term “melt curve analysis” refers to the use of the dissociation characteristics of a segment of double-stranded nucleic during heating. Originally, strand dissociation was observed using UV absorbance measurements, but techniques based on fluorescence measurements are now the most common approach. The temperature-dependent dissociation between two DNA-strands can be measured in a “melt assay,” for example, using a DNA-intercalating fluorophore, such as SYBR green or EvaGreen, or fluorophore-labelled DNA probes. In the case of SYBR green (which fluoresces 1000-fold more intensely while intercalated in the minor groove of two strands of DNA), the dissociation of the DNA during heating is measurable by the large reduction in fluorescence that results. Alternatively, juxtapositioned probes (one featuring a fluorophore and the other, a suitable quencher) can be used to determine the complementarity of the probe to the target region of nucleic acid.

[0048] A “reagent” refers broadly to any agent used in a reaction, other than the analyte (e.g., nucleic acid being analyzed). Illustrative reagents for a nucleic acid amplification reaction include, but are not limited to, buffer, metal ions, polymerase, reverse transcriptase, primers, template nucleic acid, nucleotides, labels, dyes, nucleases, dNTPs, and the like. Reagents for enzyme reactions include, for example, substrates, cofactors, buffer, metal ions, inhibitors, and activators.

[0049] The term “label,” as used herein, refers to any atom or molecule that can be used to provide a detectable and/or quantifiable signal. In particular, the label can be attached, directly or indirectly, to a nucleic acid or protein. Suitable labels that can be attached to probes include, but are not limited to, radioisotopes, fluorophores, chromophores, mass labels, electron dense particles, magnetic particles, spin labels, molecules that emit chemiluminescence, electrochemically active molecules, enzymes, cofactors, and enzyme substrates.

[0050] The term “dye,” as used herein, generally refers to any organic or inorganic molecule that absorbs electromagnetic radiation and produces a detectable signal (e.g., a fluorescent signal). [0051] The term “quencher,"’ as used herein generally refers to any organic or inorganic molecule that reduces the level of a detectable signal.

[0052] As used herein, the term “detecting” refers to “determining the presence of’ an item, such as a nucleic acid sequence, e.g., one that is indicative of the presence of a non-tuberculosis my cobacterium (NTM). Detection can include the determination of the presence of a NTM, without definitive identification of that NTM; the determination of the presence of one or more NTM belonging to a class of NTM; or the determination of the presence of a particular, known NTM strain.

[0053] The term “identifying” as used herein refers to the action of recognizing a sample as having a certain strain or species of NTM, e.g., Mycobacterium abscessus, Mycobacterium asiaticum. Mycobacterium chelonae. or Mycobacterium gastri.

[0054] As used herein, the term “treatment regimen” refers to any medical intervention intended to mitigate the symptoms and/or the pathology of a disorder. The treatment regimen can include one or more actions (e.g., bed rest, increasing fluid intake), non-prescription or prescription medications, supplements, foods, drinks, or the use of medical devices (e.g.. a respirator).

[0055] As used herein, “Clinical Laboratory Improvement Amendments (CLIA)” refers to The Clinical Laboratory' Improvement Amendments of 1988 (CLIA) regulations in effect as of the original filing date of the present application. The CLIA regulations include federal standards applicable to all U.S. facilities or sites that test human specimens for health assessment or to diagnose, prevent, or treat disease. A “CLIA-complianf ‘ test is one that complies with these regulations. “CLIA-waived” tests include tests that does not comply with all of these regulations. For example, CLIA-waived tests include test systems cleared by the U.S. Food and Drug Administration for home use and those tests approved for waiver under the CLIA criteria.

[0056] An “endogenous control,” as used herein refers to a moiety that is naturally present in the sample to be used for detection. In some embodiments, an endogenous control is a “sample adequacy control” (SAC), which may be used to determine whether there was sufficient sample used in the assay, or whether the sample comprised sufficient biological material, such as cells. In some embodiments, an endogenous control is an RNA (such as an mRNA, tRNA, ribosomal RNA, etc.), such as a human RNA for a human sample. Nonlimiting exemplary endogenous controls include ABL mRNA, GUSB mRNA, GAPDH mRNA, TUBB mRNA, and UPKla mRNA. In some embodiments, an endogenous control, such as an SAC, is selected that can be detected in the same manner as the target region (e.g., RNA) is detected and, in some embodiments, simultaneously with the target region (e.g., RNA).

[0057] An “exogenous control,” as used herein, refers to a moiety that is added to a sample or to an assay, such as a “sample processing control” (SPC). In some embodiments, an exogenous control is included with the assay reagents. An exogenous control is typically selected that is not expected to be present in the sample to be used for detection or is present at very low levels in the sample such that the amount of the moiety naturally present in the sample is either undetectable or is detectable at a much lower level than the amount added to the sample as an exogenous control. In some embodiments, an exogenous control comprises a nucleotide sequence that is not expected to be present in the sample type used for detection of the target region (e.g., RNA). In some embodiments, an exogenous control comprises a nucleotide sequence that is not known to be present in the species from whom the sample is taken. In some embodiments, an exogenous control comprises a nucleotide sequence from a different species than the subject from whom the sample was taken. In some embodiments, an exogenous control comprises a nucleotide sequence that is not known to be present in any species. In some embodiments, an exogenous control is selected that can be detected in the same manner as the target region (e.g., RNA) is detected and. in some embodiments, simultaneously with the target region (e g., RNA). In some embodiments, the exogenous control is an RNA. In some such embodiments, the exogenous control is an Armored RNA®, which comprises RNA packaged in a bacteriophage protective coat. See, e.g., WalkerPeach, et al, Clin. Chem. 45: 12: 2079- 2085 (1999).

[0058] The term “sample” as used herein includes any biological specimen obtained from a patient. Samples include, without limitation, whole blood, plasma, serum, red blood cells, white blood cells (e.g., peripheral blood mononuclear cells), ductal lavage fluid, nipple aspirate, lymph (e.g.. disseminated tumor cells of the lymph node), bone marrow aspirate, saliva, urine, stool (i.e., feces), sputum, bronchial lavage fluid, tears, fine needle aspirate (e.g., harvested by random periareolar fine needle aspiration), any other bodily fluid, a tissue sample (e.g.. tumor tissue) such as a biopsy of a tumor (e.g., needle biopsy) or a lymph node (e.g., sentinel lymph node biopsy), and cellular extracts thereof. In some embodiments, the sample is whole blood or a fractional component thereof such as plasma, serum, or a cell pellet.

[0059] The term '‘overlapping” or '‘staggered’’ hybridizing probes as used herein refers to two or more probes that are designed to span or ‘tile’ across genomic regions of interest, each having a nucleotide sequence (such as from about 2-20 nucleotides or from about 5-15 nucleotides) that is homologous with a portion of another probe located at or near its 3’ end or 5’ end. While the “overlapping” hybridizing probes can independently vary significantly in size, probes are generally designed to comprise about 20-60 nucleotides long and bind to nucleic acid template strand with an overlap “target” region of about 5-15 bp. For example, the probes can comprise about 20-60 (such as 22. 24. 26. 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, or 58) nucleotides long and bind to nucleic acid template strand with an overlap “target” region of 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 bases. The term “about” as used herein in connection with a numerical value is meant to have its usual meaning in the context of the numerical value. Where necessary the word “about” may be replaced by the numerical value ±10%. or ±5%, or ±2%. or ±1%.

[0060] The term “sequential” hybridizing probes as used herein refers to two or more non-overlapping probes that are designed to span or ‘tile’ across genomic regions of interest. The sequential hybridizing probes may be end-to-end tiled with no bases separating them or they may be spaced farther apart.

[0061] The term '‘spaced out” hybridizing probes as used herein refers to two or more non-overlapping probes that are designed to span or ‘tile’ across genomic regions of interest. The spaced-out hybridizing probes are tiled with farther apart such that one or more bases separate them when hybridized to the genomic target of interest.

[0062] The term '‘wavelength” as used herein can refer to a specific, well- defined wavelength, for example representing a dominant or peak wavelength within a range of wavelengths. The term “wavelength” can also refer to a band of wavelengths (also termed a wavelength band) which can be, for example, up to a few tens of nanometers wide emitted by a fluorophore. Accordingly, the phrase “same wavelength” as used herein can refer to a single wavelength or two or more different wavelengths within a wavelength band. Compositions and Methods

[0063] Disclosed herein are compositions and methods for maximizing the detection of one or more targets by generating unique double, triple, or more melt temperature (T_m) signatures from probes with the same detectable label (e.g., a fluorophore). This multi-signature based on a single fluorophore can be used in conjunction with unique T_m signatures from other probes with different fluorophores in a multiplex reaction system to uniquely identify many sequences, enabling high-level multiplexing. More specifically, the disclosure provides compositions and methods for detecting one or more target regions of nucleic acid sequences at a wavelength (e.g., a specifically defined wavelength or wavelength band) by adding, to a single reaction system, a plurality of oligonucleotide probes labeled with a fluorophore (or fluorophores having the same or similar detection wavelength) and designed such that melt profile (e.g., T_m value) are unique for each oligonucleotide probe hybridized to the target regions.

[0064] In one aspect, compositions and methods are provided for detecting a target region of nucleic acid at a wavelength by hybridizing with a plurality of oligonucleotide probes each having a detectable label (e g., fluorescent dye), wherein the detectable label on the plurality of oligonucleotide probes are the same or have similar detection wavelengths. The plurality of oligonucleotide probes is designed such that the melt profile (e.g., T_m value) of each oligonucleotide probes when hybridized to the target region is unique. Combining the melt profiles (e g., T_m values) generated by the plurality of probe-target hybrids result in a melt profile signature that serve as highly accurate identifier for a target region.

[0065] Preferably, the plurality of oligonucleotide probes is designed such that the melt profile signature comprises a plurality (equivalent to the number of oligonucleotide probes) of melt temperature peaks (T_m values) separated by at least 4°C. The melt profile and subsequent melt temperature (T_m) of the probe-target hybrid reflects the degree to which the oligonucleotide probe is complementary to the target region in the amplicon. Thus, in some cases, an oligonucleotide probe hybridized to a target region may not show a melt temperature peak (T_m value) and therefore, the melt profile signature may comprise melt temperature peaks (Tm values) that are less than the number of oligonucleotide probes used. For example, if two oligonucleotide probes are used, the melt profile signature may comprise one or two melt temperature peaks (T_m values), depending on the probe sequence and/or target region of nucleic acid. A melt profile signature with only one Tm value is still representative of a signature. [0066] FIG. 1 provides exemplary compositions and methods using two oligonucleotide probes and include a first oligonucleotide probe, labelled as “Probe 1” and hybridized to a first subsequence of a target region of interest and second oligonucleotide probe, labelled “Probe 2” and hybridized to a second subsequence of the target region of interest. The first oligonucleotide probe (Probe 1) and second oligonucleotide probe (Probe 2) are demonstrated to be labelled with the same fluorescent dye (CF5) which emits light at the same wavelength. Although not depicted, the first oligonucleotide probe and second oligonucleotide probe may be labelled with two or more fluorescent dyes.

[0067] In another aspect of the disclosure, compositions and methods are provided for simultaneously detecting a plurality of target regions at a wavelength (e.g., a specifically defined wavelength or wavelength band). Particularly, the method can include simultaneously detecting a plurality of target regions of nucleic acid at a wavelength by hybridizing a plurality of oligonucleotide probes (each having a detectable label e g., fluorescent dye) to each target region, wherein the detectable labels on the plurality of oligonucleotide probes are the same or have similar detection wavelengths. To reduce the number of probes in the reaction mixture, the methods can use sloppy molecular beacon probes (SMB). Sloppy molecular beacon probes possess relatively long probe sequences, enabling them to form hybrids with amplicons from many different target regions (e.g., different specie) despite the presence of mismatched base pairs. Sloppy molecular beacons can generate unique melt profiles (including T_m values) for target nucleic acid sequences that differ by as little as one nucleotide. Thus, the simultaneous use of a set of sloppy molecular beacons (two or more SMB probes), each possessing a different probe sequence and each labeled with the same colored fluorophore can be used to provide multiple sets of melt profiles (e.g., T_m values), each serving as a unique, target region-specific signature. As further described herein, the methods are not limited to the use of SMB probes. As described herein, the plurality of oligonucleotide probes is designed such that the melt profile (e.g., T_m value) of each oligonucleotide probe when hybridized to the target region is unique.

[0068] In further aspects of the disclosure, compositions and methods are provided for simultaneously detecting a plurality of target regions at more than one wavelengths, wherein each target region of nucleic acid is hybridized to a plurality of oligonucleotide probes having detectable labels that are the same or have similar detection wavelengths. For example, the method can include simultaneously detecting a first and second target region of nucleic acid, comprising hybridizing the first target region to a plurality of oligonucleotide probes having detectable labels that emit light at a first wavelength, and hybridizing the second target region to a plurality of oligonucleotide probes having detectable labels that emit light at a second wavelength, different than the first wavelength. Any suitable probes, including SMB probes, linear probes, or combinations thereof, can be used in the disclosed methods.

Target region of nucleic acids

[0069] The present disclosure provides compositions and methods aimed at detecting and optionally identifying via melt profile signature, a plurality of target regions of nucleic acids. The compositions and methods provided can simultaneously detect and optionally identify' via melt profile signature, at least 2 target regions, at least 3 target regions, at least 4 target regions, at least 5 target regions, at least 6 target regions, at least 7 target regions, at least 8 target regions, at least 9 target regions, at least 10 target regions, at least 12 target regions, at least 14 target regions, at least 15 target regions, at least 16 target regions, at least 18 target regions, or at least 20 target regions of nucleic acids. In some embodiments, the compositions and methods provided can simultaneously detect and optionally identify via melt profile signature, up to 5 target regions, up to 10 target regions, up to 15 target regions, up to 20 target regions, up to 25 target regions, up to 30 target regions, up to 35 target regions, up to 40 target regions of nucleic acids, and any value in between (e.g., up to 32 target regions). The detectable label (e.g., fluorophore) used to generate the melt signatures for the plurality of target regions can be the same or different. The number of different wavelengths detected is dependent on the limitation of the instrument used. For example, a seven-color multiplex system is described, e.g., in Lee, et al., BioTechniques, 27: 342-349 and a ten-color multiplex system has been described, e.g., in Xie. et al.. N Engl J Med 2017; 377: 1043-1054 and Chakravorty, et al., J Clin Microbiol 2016; 55: 183-198. Accordingly, the method can comprise generating and detecting melt profile signatures for the plurality of target regions at up to 4 different wavelengths, up to 6 different wavelengths, up to 10 different wavelengths, and any value in between (e.g.. up to 9 different wavelengths). In some embodiments, melt profile signature(s) for each target region can be generated and detected at a single wavelength or at multiple wavelengths.

[0070] As described herein, a target region is a segment of nucleic acid which can be representative of the presence of a gene, a species or organism, and/or a class or organisms. In some embodiments, the target region of nucleic acid can be selected from pathogenic nucleic acids, bacterial nucleic acids, viral nucleic acids, fungal nucleic acids, parasite nucleic acids, protozoa nucleic acids, or a combination thereof. In certain embodiments, the target region of nucleic acid can be selected from a Mycobacterium tuberculosis gene, for example, an antimicrobial resistance Mycobacterium tuberculosis gene, an antimicrobial susceptible Mycobacterium tuberculosis gene, or a nontuberculous mycobacterium (NTM). or a combination thereof. In some examples, the target region of nucleic acid includes a nontuberculous mycobacterium.

[0071] In more specific examples, the target region of nucleic acid can include one or more infectious agents. For example, the target region of nucleic acid can include a viral infectious agents such as the HIV, SIV, FIV, VHC, VHB, VHA, VHE virus, the VZV, CMV. EBV, VHS1. VHS2 virus; a bactenal infectious agent such as Mycobacterium tuberculosis, Mycobacterium bovis, Mycobacterium leprae, Borrelia burgdorferi stricto sensu, Borrelia afzelii, Borrelia garinii, Borrelia spielmanii, Clostridium difficile, Clostridium botulinum, Salmonellas, Klebsiella, Legionella, Proteus, Klebsiella, Escherichia coll, Shigella, Pseudomonas aeruginosa, Staphylococcus aureus. Treponema pallidum, a yeast such as Candida albicans, a fungal infectious agent such as Aspergillus fumigatus, Mucorales, and/or a protozoan infectious agent such as Leishmania, Trichomonas vaginalis, Plasmodium.

[0072] In some examples, the target region of nucleic acids can be selected from a pathogenic viral infection selected from astrovirus, coronavirus (e.g., a- coronavirus, -coronavirus, or SARS-CoV-2), multisystem inflammatory syndrome, dengue, influenza, influenza A, influenza B, metapneumovirus, rhinovirus, Zika, adenovirus, Chlamydia pneumoniae, enterovirus, mycoplasma, Bordetella spp., parainfluenza, respiratory syncytial virus (RSV), or any combination thereof. In some examples, the target region of nucleic acids is selected from a respiratory pathogen. [0073] In some examples, the target region of nucleic acids is selected from a gastrointestinal pathogen. This selection can include a combination of bacterial, viral and parasitic organisms. In some examples, the target region of nucleic acids includes Campylobacter, STEC stxl, STEC stx2. Salmonella, Shigella/EIEC, Yersinia enterocolitica, Vibrio cholerae, Vibrio parahaemolyticus. Cryptosporidium, Giardia lamblia, Norovirus, and combinations thereof.

[0074] In some examples, the target region of nucleic acids can be selected from a pathogenic bacterial infection such as Acinetobacter, Aerococcus, Bacillus, Bacteriodes, Borrelia, Clostridium, Enterobacter, Enterococcus, Escherichia, Klebsiella, Mycobacterium, Neisseria. Pseudomonas. Serratia, Staphylococcus, Streptococcus or any combination thereof. For example, the target region of nucleic acids can include Borrelia burgdorferi, Borrelia mayonii, Mycobacterium tuberculosis, or any combination thereof.

[0075] In some examples, the target region of nucleic acids can be selected from at least one bacteria selected from Mycobacterium tuberculosis, Mycobacterium africanum, Mycobacterium bovis, Mycobacterium caprae, Mycobacterium microti, Mycobacterium leprae, Mycobacterium lepromatosis, Mycobacterium avium, Mycobacterium silvaticum, Mycobacterium hominissuis, Mycobacterium paratuberculosis, Mycobacterium kansasii, Mycobacterium xenopi, Mycobacterium simiae, Mycobacterium abcessus, Mycobacterium fortuitum, Mycobacterium chelonae, Mycobacterium ulcerans, Mycobacterium marinum and/ or Mycobacterium fortuitum. In some examples, the target region of nucleic acids can be selected from at least one non-tuberculous mycobacteria (NTM). As used herein and throughout the entire description, the term “non-tuberculous mycobacteria" means mycobacteria which do not cause tuberculosis or leprosy. In some embodiments the non-tuberculosis bacteria are selected from a group consisting of Mycobacterium avium, Mycobacterium silvaticum, Mycobacterium hominissuis. Mycobacterium paratuberculosis, Mycobacterium kansasii, Mycobacterium xenopi, Mycobacterium simiae.

Mycobacterium abcessus, Mycobacterium fortuitum, Mycobacterium chelonae, Mycobacterium ulcerans, Mycobacterium marinum, Mycobacterium gordonae and/or Mycobacterium fortuitum. In some examples, the target region of nucleic acid can include Mycobacterium avium complex (MAC), including bacteria selected from the group consisting of Mycobacterium avium. Mycobacterium silvaticum, Mycobacterium hominissuis and Mycobacterium paratuberculosis. In some embodiments the non-tuberculosis bacteria are selected from a group consisting of Mycobacterium abscessus. Mycobacterium avium, Mycobacterium chelonae. Mycobacterium fortuitum, Mycobacterium gordonae, Mycobacterium intracellulare, Mycobacterium kansasii, Mycobacterium simiae, Mycobacterium xenopi, or a combination thereof.

[0076] In some examples, the target region of nucleic acids is selected from a Mycobacterium tuberculosis gene. This selection can include an antimicrobial resistance Mycobacterium tuberculosis gene, an antimicrobial susceptible Mycobacterium tuberculosis gene (e.g., RIF susceptibility ), a nontuberculous mycobacterium, a wild-type Mycobacterium tuberculosis gene, or a combination thereof. The antimicrobial resistance Mycobacterium tuberculosis gene can be selected from isoniazid (INH) resistance, fluoroquinolone (FLQ) resistance, ethionamide (ETH) resistance, rifampicin resistance, amikacin (AMK) resistance, capreomycin (CAP) resistance, kanamycin (KAN) resistance, aminoglycoside resistance, bedaquiline resistance, clofazimine resistance, delamanid resistance, ethambutol resistance, linezolid resistance, pyrazinamide resistance, streptomycin resistance, or a combination thereof. In some examples, the target region of nucleic acid can be selected from the rpoB RRDR gene. rpoB 491 gene. rpoB V170 gene, IS6110 gene, IS 1081 gene, fabGl gene, inhA promoter, katG gene, gyrA gene, gyrB gene, pncA gene, rplC gene, rrl gene, rrs gene, atpE gene, ahpC gene, eis promoter, oxyR-ahpC (ahpC) intergenic region, or Rv0678 gene of Mycobacterium tuberculosis.

[0077] In some examples, the methods disclosed here can be used to detect target region of nucleic acids in a respiratory panel for detecting upper respiratory infections, a blood culture panel for detecting blood stream infections, a gastrointestinal panel for detecting GI infections, or a meningitis panel for detecting cerebrospinal fluid infections.

[0078] In some examples, the methods disclosed here can be used to detect target region of nucleic acids for viral or bacterial pathogens selected from a novel coronavirus, hantavirus, cytomegalovirus, coxsackie virus, herpes simplex virus, echo virus, influenza virus C, Streptococcus pneumoniae, Moraxella catarrhalis, Haemophilus influenzae, Haemophilus parainfluenzae, a group A streptococcus. Streptococcus pyogenes, Klebsiella pneumoniae, a. Pseudomonas species, a Neisseria species, Histoplasnia capsulatum, Coccidioides immitis, Blastomyces dermatitidis, Paracoccidioides brasiliensis, a Candida species, an Aspergillus species. aMucor species, Cryptococcus neoformans, and/ or Pneumocystis carinii. [0079] Bioinformatic analysis of multiple data bases can be earned out to identify primers and probes for highly conserved regions in the genomes of these pathogens.

Multiplex Reaction

[0080] The target region of nucleic acids discussed herein can be detected by nucleic acid amplification in an assay, particularly, in multiplex amplification reactions, which can be designed to detect 2 or more, 3 or more. 4 or more, 5 or more, 6 or more.

7 or more, 8 or more, 9 or more, 10 or more, 11 or more, 12 or more, 13 or more, 14 or more, 15 or more, 16 or more, 17 or more, 18 or more, 19 or more, 20 or more, 21 or more, 22 or more, 23 or more, 24 or more, 25 or more, 26 or more, 27 or more, 28 or more, 29 or more, 30 or more. 31 or more, 32 or more, 33 or more, 34 or more, 35 or more, 36 or more, 37 or more, 38 or more, 39 or more, or 40 or more target regions of nucleic acids per amplification reaction mixture. This degree of multiplexing can be achieved by a method of generating and detecting unique melt profile (e.g., melt temperature) signatures for each target. As described herein, the method uses two or more probes that hybridizes to a target region of nucleic acid, wherein both probes comprise the same fluorophore and quencher options.

[0081] Recognized herein are various issues with currently available multiplexed PCR methods. For instance, while multiplexing a large number of target amplification reactions (e.g.. multiplexed PCR) may be possible, it is not straightforward to detect multiple amplicons simultaneously. So far, multiplexed q- PCR methods, defined as the processes by which one amplifies and detects a plurality of nucleic acid sequences simultaneously in a single reaction chamber, have been implemented for a small number of amplicons, generally less than ten. It is of great interest to efficiently multiplex the assays in the same reaction volume and allow for multiple concurrent target amplification and detection in the same reaction chamber. Such an approach may not only better utilize the original nucleic acid sample, but also significantly reduce any complexities associated with the fluidics and liquid-handling procedures for running multiple single-plex reactions.

[0082] Attempts at creating multiplexed q-PCR methods have been plagued by practical issues of simultaneously detecting different nucleic acid sequences in a single sample. A conventional approach is to associate different reporter molecules (e.g.. fluorescent dyes) to individual amplicons during the PCR reaction which may enable parallel detection of individual reporters by different ‘‘colors’'. While such approach, in theory, may offer parallelism, it is limited by: (i) the number of different reporter molecules available; and (ii) the availability of imagers and detectors capable of differentiating different signals. Another conventional approach to offer multiplexing capability' is to divide the biological sample of interest and physically place it, using fluidic systems, into separate, single and isolated amplification chambers. While this approach may effectively create multiplex q-PCR by performing multiple single-plex (i.e., one amplicon per chamber) q-PCR reactions, it may be suboptimal, since it may reduce the number of target region of nucleic acids in each chamber which may create stochastic anomalies (Poisson noise) in the acquired data when the original sample has a small concentration. Further, it requires complex fluidic handling procedures.

[0083] Highly-multiplexed detection of DNA sequences in a sample may be done through adopting analytical platforms such as DNA microarrays or nextgeneration DNA sequencers. Microarrays, in particular, are massively-parallel, affinity-based biosensors where target regions are captured selectively from the same sample at different addressable coordinates (e.g., pixels) on a solid surface. Each addressable coordinate can have a unique capturing DNA or RNA probe, complementary to a target region of nucleic acid to be detected in the sample. While microarrays may offer high multiplexing capability, they are semiquantitative and are inferior in terms of limit-of-detection (LOD) and detection dynamic range (DDR), due to their end-point detection nature (i.e., no real-time detection) and the fact that they lack any target amplification.

[0084] The methods described herein increases the number of target region of nucleic acids detected per optical channel by generating and detecting unique melt profile signatures for each target region. The melt profile (e.g., melt temperature) window for each target region can be designed such that it is dependent on the sequence variation of the target. Exemplified herein are several channel options and designs for each target to find the optimum arrangement. As an example, two or more of AT abscessus, M. avium paraTB, M. chelonae, M. fortuitum, M. gordonae. M. intracellulare, M. kansasii, M. imiae. and AT. xenopi targets can be detected in one optical channel using the same plurality of probes. The plurality of probes used various levels of mismatches based on the particular species (e.g., from about 70% to 100% complementarity among the species) resulting in detection by melt at different melt temperatures.

[0085] In addition, to increase the number of target regions detected per optical channel, TaqMan® Gene Expression Assays and melt probes can be combined in the same optical channel using melt probe with a T_m below annealing temperature (no amplification curve). In some instances, due to the high mutation rate of some viral target organisms, which make it challenging to find conserved regions, amplification detection can be used for viral organisms to avoid a large variation in the melt probes and T_m windows associated with the mutations. Accordingly, bacterial and other targets can be detected using the melt temperature detection methods disclosed herein.

[0086] One advantage of the methods disclosed herein is a reduction in the number of oligonucleotides combined in one reaction mixture, thereby reducing unwanted interactions between them. In some embodiments, modified nucleotides can be used in the reaction mixture to further reduce and eliminate primer-primer interactions, if any.

[0087] Exemplary primers and probes for NTM genes being targeted in an illustrative embodiment of the methods herein are provided below in the section titled Examples. Particular embodiments of a method may include detection of a unique melt temperature signature for each target region in a single optical channel. Other particular embodiments of a method disclosed herein can include detection of unique melt temperature signatures for each target region in at least two optical channels.

[0088] Other considerations for primers and probes for detecting the target region of nucleic acids are described in more detail below' in the section entitled ‘“Exemplary Polynucleotides.”

Controls

[0089] In some embodiments, an assay described herein comprises detecting the target region of nucleic acids described above and at least one endogenous control. In some embodiments, the endogenous control is a sample adequacy control (SAC). In some such embodiments, if no target region of nucleic acid is detected in a sample, and the SAC is also not detected in the sample, the assay result is considered '‘invalid” because the sample may have been insufficient. While not intending to be bound by any particular theory, an insufficient sample may be too dilute, contain too little cellular material, or contain an assay inhibitor, etc. In some embodiments, the failure to detect the SAC may indicate that the assay reaction failed. In some embodiments, an endogenous control is an RNA (such as an mRNA, tRNA, ribosomal RNA, etc.). Nonlimiting exemplary endogenous controls include ABL mRNA, GUSB mRNA, GAPDH mRNA, TUBB mRNA, and UPKla mRNA.

[0090] In some embodiments, an assay described herein comprises detecting the target region of nucleic acids described above and at least one exogenous control. In some embodiments, the exogenous control is a sample processing control (SPC). In some such embodiments, if no target region of nucleic acids described above is detected in a sample, and the SPC is also not detected in the sample, the assay result is considered “invalid’' because there may have been an error in sample processing, including but not limited to, failure of the assay. Nonlimiting exemplary errors in sample processing include inadequate sample processing, the presence of an assay inhibitor, the presence of a nuclease (such as an RNase), or compromised reagents, etc. In some embodiments, an exogenous control (such as an SPC) is added to a sample. In some embodiments, an exogenous control (such as an SPC) is added during performance of an assay, such as with one or more buffers or reagents. In some embodiments, when a GeneXpert® system is to be used, the SPC is included in the GeneXpert® cartridge. In some embodiments, an exogenous control (such as an SPC) is an Armored RNA®. which is protected by a bacteriophage coat.

[0091] In some embodiments, an endogenous control and/or an exogenous control is/are detected contemporaneously, such as in the same assay, as detection of the target region of nucleic acids. In some embodiments, an assay comprises reagents for detecting the target region of nucleic acids described above, and a SAC and/or an exogenous control, simultaneously in the same assay reaction mixture. In some such embodiments, for example, an assay reaction mixture comprises primer sets for amplifying the target region of nucleic acids described above, a primer set for amplifying a SAC and/or a primer set for amplifying an exogenous control, as well as optional labeled probes for detecting the amplification products (such as, for example, TaqMan® probes).

Polynucleotides

[0092] Polynucleotides are provided for detecting the target region of nucleic acids described above. In some embodiments, synthetic polynucleotides are provided. Synthetic polynucleotides, as used herein, refer to polynucleotides that have been synthesized in vitro either chemically or enzymatically. Chemical synthesis of polynucleotides includes, but is not limited to, synthesis using polynucleotide synthesizers, such as OligoPilot™ (GE Healthcare), ABI 3900 DNA Synthesizer (Applied Biosystems), and the like. Enzymatic synthesis includes, but is not limited, to producing polynucleotides by enzy matic amplification, e.g., PCR. A polynucleotide may comprise one or more analog of the canonical nucleotides (e.g., modified nucleotides).

[0093] In some embodiments, a polynucleotide is provided that comprises a region that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 87%, 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% identical to, 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% complementary to, 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. at least 28, at least 29, at least 30, at least 32, at least 34, at least 35, at least 36, at least 38, at least 40, at least 42, at least 44, at least 45, at least 46, at least 48, at least 50, at least 52, at least 54, at least 55, at least 56, at least 58, at least 59, or at least 60 contiguous nucleotides of the target region of nucleic acids, and/or exemplary controls discussed above.

[0094] In various embodiments, an 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 various embodiments, a polynucleotide comprises fewer than: 200, 150, 100, 50, 40, 30, or 20 nucleotides. In various embodiments, an exemplary polynucleotide is between about 6 and 200, between about 8 and 200, between about 8 and 150, between about 8 and 100, between about 8 and 75, between about 8 and 50, between about 8 and 40, between about 8 and 30. between about 15 and 100, between about 15 and 75. between about 15 and 50, between about 15 and 40, or between about 15 and 30 nucleotides long. In other embodiments, an exemplary SMB polynucleotide is between about 30 and 200, between about 40 and 200, between about 40 and 150, between about 40 and 100, between about 40 and 75. or between about 40 and 50 nucleotides long. Excluding the stem region of the SMB polynucleotide, the SMB polynucleotide may include about 15 or less, about 12 or less, about 10 or less, about 8 or less, about 6 or less, about 5 or less, about 4 or less, from about 4-15, from about 4-12, or from about 4-10 mismatches with the target region of nucleic acids. [0095] In some embodiments, detection of each target region of nucleic acid can be carried out using two or more probes specific for each target region, wherein the two or more probes have the same or similar detectable label. By using probes labeled with the same or similar detectable moieties (e.g.. same fluorescent reporter dyes), numerous target regions can be detected simultaneously in a single reaction mixture and in a single optical channel by melt temperature detection. 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 target region of nucleic acids can be used in a single reaction mixture and detected in a single optical channel. In some embodiments, the method employs probes labeled with different detectable moieties (e.g., different fluorescent reporter dyes), so numerous target regions can be detected simultaneously in a single reaction mixture but in more than one optical channel. 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 can be used in a single reaction mixture. Whether a single optical channel (single detectable label) or more than one optical channel (two or more detectable labels) are used, the methods disclosed herein provides that each target region can be independently monitored using the disclosed multiplexing technology. In some embodiments, real time amplification curves may be generated to characterize additional target region of nucleic acids that each use the same label as melt detection, but such analysis may not be necessarily required.

Polynucleotide Modifications

[0096] In some embodiments, the methods of detecting at least one target region described herein employ one or more polynucleotides that have been modified, such as polynucleotides comprising one or more affinity-enhancing nucleotide analogs. Modified polynucleotides useful in the methods described herein include primers for reverse transcription, PCR amplification primers, and probes. In some embodiments, the incorporation of affinity-enhancing nucleotides increases the binding affinity and specificity of a polynucleotide for its target region as compared to polynucleotides that contain only the canonical deoxyribonucleotides, which allows for the use of shorter polynucleotides or for shorter regions of complementarity between the polynucleotide and the target region. [0097] In some embodiments, affinity-enhancing nucleotide analogs include nucleotides comprising one or more base modifications, sugar modifications, and/or backbone modifications. In some embodiments, modified bases for use in affinityenhancing 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, affinity-enhancing nucleotide analogs include nucleotides having modified sugars such as 2'-substituted sugars, such as 2'-O-alkyl-ribose sugars, 2'- amino-deoxyribose sugars, 2'-fluoro-deoxyribose sugars, 2'-fluoro-arabinose sugars, and 2'-O-methoxyethyl-ribose (2'MOE) sugars. In some embodiments, modified sugars are arabinose sugars, or d-arabino-hexitol sugars.

[0098] In some embodiments, affinity-enhancing nucleotide analogs include backbone modifications such as the use of peptide nucleic acids (“PNA”) (e.g., an oligomer including nucleobases linked together by an amino acid backbone). Other backbone modifications include phosphorothioate linkages, phosphodiester-modified nucleic acids, combinations of phosphodiester and phosphorothioate nucleic acid, methyl phosphonate, alkylphosphonates, phosphate esters, alkylphosphonothioates, phosphoramidates, carbamates, carbonates, phosphate triesters, acetamidates, carboxymethyl esters, methylphosphorothioate, phosphorodithioate, p-ethoxy modifications, and combinations thereof.

[0099] In some embodiments, a polynucleotide includes at least one affinityenhancing nucleotide analog that has a modified base, at least nucleotide (which may be the same nucleotide) that has a modified sugar, and/or at least one intemucleotide linkage that is non-naturally occurring.

[0100] In some embodiments, an affinity -enhancing nucleotide analog contains a locked nucleic acid (“LNA”) sugar, which is a bicyclic sugar. In some embodiments, a polynucleotide for use in the methods described herein comprises one or more nucleotides having an LNA sugar. In some embodiments, a polynucleotide contains one or more regions consisting of nucleotides with LNA sugars. In other embodiments, a polynucleotide contains nucleotides with LNA sugars interspersed with deoxyribonucleotides. See, e.g., Frieden, M., et al., (2008) Curr. Pharm. Des.

14(11):1138-1142. Primers

[0101] In some embodiments, the polynucleotide is a primer. Primers useful in the methods described herein are generally capable of selectively hybridizing to: genomic DNA, a target RNA (genomic or transcript), a cDNA reverse transcribed from the target RNA, and/or an amplicon that has been amplified from genomic DNA, a target RNA, or a cDNA (collectively referred to as "‘template”), and, in the presence of the template, a polymerase and suitable buffers and reagents can be extended to form a primer extension product. Primers are generally of a sufficient length to ensure selective hybridization to their target regions. Generally, primers of at least 15 nucleotides in length hybridize specifically in most contexts, and this length can be reduced, e.g., by including of affinity-enhancing modifications, such as those discussed above. Primers can but need not be exactly complementary to their target regions. Primers can have any degree of complementarity described above for exemplary polynucleotides. In illustrative embodiments, primers can be about 8 to about 40 nucleotides in length and at least 90% complementary’ to their target regions; about 8 to about 40 nucleotides in length and at least 95% complementary to their target regions; about 8 to about 40 nucleotides in length and at least 99% complementary to their target regions; about 8 to about 30 nucleotides in length and at least 90% complementary' to their target regions: about 8 to about 30 nucleotides in length and at least 95% complementary to their target regions; and/or about 8 to about 30 nucleotides in length and at least 99% complementary to their target regions. In embodiments wherein a primer is less than 100% complementary to it target region, having the 3’ nucleotide in the primer be complementary' to its target region facilitates the production of an extension product.

[0102] In some embodiments, a primer that selectively hybridizes to its target region hybridizes to its target region with at least 5 -fold greater affinity' than to nontarget region under the same assay conditions. In some embodiments, a primer that selectively hybridizes to its target region hybridizes to its target region with at least 10- fold greater affinity than to non-target region under the same assay conditions.

[0103] In some embodiments, a primer pair is designed to produce an amplicon that is about 50 to about 1500 nucleotides long, about 50 to about 1000 nucleotides long, about 50 to about 750 nucleotides long, about 50 to about 500 nucleotides long, about 50 to about 400 nucleotides long, about 50 to about 300 nucleotides long, about 50 to about 200 nucleotides long, about 50 to about 150 nucleotides long, about 100 to about 300 nucleotides long, about 100 to about 200 nucleotides long, about 100 to about 150 nucleotides long, and any value in between (e.g., about 86 to about 147 nucleotides long).

[0104] In some embodiments, the primer is labeled with a detectable moiety⁷. In some embodiments, a primer is not labeled.

Probes

[0105] In some embodiments, the polynucleotide is a probe. Probes useful in the methods described herein are generally capable of selectively hybridizing to: genomic DNA, a target RNA (genomic or transcript), a cDNA reverse transcribed from the target RNA, and/or an amplicon that has been amplified from genomic DNA, a target RNA, or a cDNA (collectively referred to as '’template"). The probes used in the methods disclosed herein can be selected from a linear probe, a molecular beacon probe, a Scorpion probe, or a combination thereof.

[0106] As disclosed herein, the probes (e.g., sloppy molecular beacons) are used in combinations of two or more to detect a target region of nucleic acid. Because the two or more probes differ in their nucleic acid sequences, their relative affinities for the target region (or even for different target regions) are different. For example, a first probe may bind strongly to a first target region, moderately to a second target region, weakly to a third target region, and not at all to a fourth target region; while a second probe may bind weakly to the first target region, strongly to the second target region, not at all to the third target region, and moderately to the fourth target region. In another example, a first probe may bind strongly to a first target region, moderately to a second target region, weakly to a third target region, and not at all to a fourth target region; while a second probe may bind weakly to the first target region and the second target region, and moderately to the third target region and the fourth target region. Additional probes will exhibit different binding patterns due to their different target region of nucleic acids. Thus, unique melt temperatures detected from combinations of probes define different target region of nucleic acids (e.g., microbial strains or species). [0107] The disclosed methods are based on deconvoluting the melt peak profile (e.g., melt temperatures) of a set of fully and/or partially hybridizing probes (such as sloppy molecular beacon probes), each labeled with a fluorophore that emits light with the same or similar wavelength optimum. [0108] Preferably, the probes (e.g., molecular beacon probes) useful in the methods disclosed herein will hybridize to more than one target region of nucleic acids. Accordingly, sloppy molecular beacons are preferred. In some examples, the two or more probes used to detect a target region of nucleic acid include a sloppy molecular beacon probe. The sloppy molecular beacon probes are known as soppy probes by virtue of their ability to bind to more than one (e.g., 2, 3, 4, 5. 6, 7, 8, 9, 10, 11, 12. 15. 20. 30. 40. 100, 1000, and any value in between, e.g.. 832) targets (e.g., variants of a given target sequence), and can be used in assays to detect the presence of one target region of nucleic acid of interest from among a number of possible sequences or even to detect the presence of two or more target regions of a nucleic acid sequence.

[0109] Conventionally, four different sloppy molecular beacons are generally insufficient to resolve a large number and variety of target sequences with high precision. Particularly, the number of different sloppy molecular beacons that can be used simultaneously in the same assay w ell is limited by the ability to resolve the emission spectrum of each fluorophore. One of the factors that may limit the sensitivity of detection by fluorescence is that the optimal emission wavelength of most fluorophores is only a few nanometers longer than their optimal excitation wavelength (Stokes shift). As a consequence of this, a portion of the excitation light reaches the detector by processes such as scattering and reflection, contributing to a background signal that limits the sensitivity. Monochromatic light sources, such as lasers, are often used to minimize the extent to which the excitation light reaches the detector. However, this prevents the use of a large number of different fluorophores in the same solution, because these light sources excite some fluorophores very well but excite other fluorophores not as well or not at all. The present disclosure provides generation and detection of unique melt temperature signatures at a wavelength (a specifically defined wavelength or w avelength band) for a large number of target region of nucleic acids in the same solution. Accordingly, the problem of using a large number of different fluorophores in the same solution is eliminated.

[0110] Generally, probes of at least 15 nucleotides in length hybridize specifically in most contexts, and this length can be reduced, e.g., by inclusion of affinity-enhancing modifications, such as those discussed above. As described herein, probes can but need not be exactly complementary to their target regions. For example, probes can deliberately include "‘mismatches” to adjust the T_m of a melt probe. Probes can have any degree of complementarity described above for exemplary' polynucleotides. In illustrative embodiments, probes can be about 8 to about 60 nucleotides in length and at least 60% complementary' to their target regions; probes can be about 8 to about 60 nucleotides in length and at least 70% complementary to their target regions; probes can be about 8 to about 60 nucleotides in length and at least 80% complementary to their target regions; probes can be about 8 to about 40 nucleotides in length and at least 70% complementary’ to their target regions; about 8 to about 40 nucleotides in length and at least 75% complementary⁷ to their target regions; about 8 to about 40 nucleotides in length and at least 80% complementary⁷ to their target regions; about 8 to about 40 nucleotides in length and at least 85% complementary to their target regions: about 8 to about 40 nucleotides in length and at least 90% complementary to their target regions; about 8 to about 40 nucleotides in length and at least 95% complementary to their target regions; about 8 to about 40 nucleotides in length and at least 99% complementary' to their target regions; about 8 to about 30 nucleotides in length and at least 90% complementary to their target regions; about 8 to about 30 nucleotides in length and at least 95% complementary to their target regions; about 8 to about 30 nucleotides in length and at least 99% complementary⁷ to their target regions. In embodiments wherein a probe is less than 100% complementary to a target region, any points or regions of non-complementarity are typically located so as not to disrupt the ability of the probe to selectively hybridize to its target region.

[OHl] In some embodiments, the probe selectively hybridizes to its target region with at least 5-fold greater affinity than to non-target region under the same assay conditions. In some embodiments, the probe selectively hybridizes to its target region hybridizes to its target region with at least 10-fold greater affinity than to nontarget region under the same assay conditions.

[0112] Persons skilled in the art can readily prepare probes. For random coil (or “linear”) probes such as TaqMan® probes, the length of the probe region complementary to intended targets is increased sufficiently that the probe binds not only to perfectly matched targets but also to targets differing, as need arises, by one or several nucleotides. For molecular beacon probes, the length of the probe region is increased but the length of the arms hybrid is kept short. Loop sequences in the range of about 25 to about 50 nucleotides in length and arms hybrids in the range of about 4 to about 6 nucleotides in length is generally satisfactory to provide an excellent starting point for probe design. A limited number of probes could be used as probes to identify many different possible target sequences in a real-time PCR reaction.

[0113] As described herein, detection of each target region utilizes two or more probes having the same or similar detectable label. The two or more probes for a target region can comprise overlapping (staggered) hybridizing probes, sequential hybridizing probes, spaced out hybridizing probes, or a combination thereof. The two or more probes can include a molecular beacon probe, a linear probe, a FRET (TaqMan®) probe, or a combination thereof. In some examples, the two or more probes can include at least two sloppy molecular beacon probes, at least two linear probes, or a sloppy molecular beacon probe and a linear probe.

Polynucleotide Labels

[0114] The probes (and in some cases the primer) is labeled with a detectable moiety. Detectable moieties include directly detectable moi eties, such as fluorescent dyes, and indirectly detectable moieties. such as members of binding pairs. When the detectable moiety is a member of a binding pair, in some embodiments, the probe can be detectable by incubating the probe with a detectable label bound to the second member of the binding pair. In some embodiments, a primer or probe is not labeled, such as when a primer or probe is immobilized, e.g., on a microarray or bead. A labeled primer is extendable, e.g., by a polymerase. In some embodiments, a probe is extendable. In other embodiments, a probe is not extendable. The following discussion centers on probes, as these are more ty pically employed for detecting in the methods described here, but those of skill in the art appreciate that the polynucleotide labeling strategies described below apply equally to the labeling of primers.

[0115] In some embodiments, the probe is a FRET probe that is labeled at the 5 '-end with a fluorescent dye (donor) and at the 3 '-end with a quencher (acceptor), and a chemical group that absorbs (i.e., suppresses) fluorescence emission from the dye when the groups are in close proximity (e.g., attached to the same probe). Thus, 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.

[0116] Illustrative FRET probes, which include, but are not limited to, a TaqMan® probe, a Molecular beacon probe and a Scorpion probe. A TaqMan® probe is a linear probe that ty pically has a fluorescent dye covalently bound at one end of the DNA and a quencher molecule covalently bound elsewhere, such as at the other end of the DNA. The FRET probe comprises a sequence that is complementary to a region of the cDNA or amplicon such that, when the FRET probe is hybridized to the cDNA or amplicon, the dye fluorescence is increased due to increased distance between dye and quencher; when the FRET probe is non-hybridized, 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 produces a fluorescence signal. In some embodiments, the amount of target nucleic in the sample is proportional to the amount of fluorescence measured during amplification.

[0117] Like TaqMan® probes, Molecular Beacons use FRET to detect a PCR product via a probe having a fluorescent dye and a quencher attached at the ends of the probe. Unlike TaqMan® probes, Molecular Beacons remain intact during the PCR cycles. Molecular Beacon probes form a stem-loop structure when free in solution, thereby allowing the dye and quencher to be in close enough proximity to cause fluorescence quenching. When the Molecular Beacon hybridizes to a target region, the stem-loop structure is abolished so that the dye and the quencher become separated in space and the dye fluoresces. Molecular Beacons are available, e.g., from Gene Link™ (see www. genelink, com/ne wsi te/ products/ mbintro . asp) .

[0118] In some embodiments. 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 hybridized to a target region. However, unlike Molecular Beacons, a Scorpion probe achieves 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 elsewhere, such as to the 3 ’-end. The 3’ portion of the probe is complementary' to the extension product of the PCR primer, and this complementary⁷ portion is linked to the 5 ‘-end of the probe by a non-amplifiable moiety. After the Scorpion primer is extended, the target-specific sequence of the probe binds to its complement within the extended amphcon. thus opening up the stemloop structure and allowing the dye on the 5 ’-end to fluoresce and generate a signal. Scorpion probes are available from, e.g., Premier Biosoft International (see www.premierbiosoft.com/tech_notes/Scorpion.html). [0119] In some embodiments, labels that can be used on the FRET probes include colorimetric and fluorescent dyes, such as Alexa Fluor dyes; BODIPY dyes, such as BODIPY FL, Cascade Blue, and Cascade Yellow; coumarin and its derivatives, such as 7-amino-4-methyl coumarin, aminocoumarin and hydroxy coumarin; cyanine dyes, such as Cy3 and Cy5; eosins and erythrosins; fluorescein and its derivatives, such as fluorescein isothiocyanate; macrocyclic chelates of lanthanide ions, such as Quantum Dye™; 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 heterodimer; and TOT AB.

[0120] Specific examples of dyes include, but are not limited to, those identified above and the following: Alexa Fluor 350. Alexa Fluor 405. Alexa Fluor 430, Alexa Fluor 488, Alexa Fluor 500. Alexa Fluor 514, Alexa Fluor 532, Alexa Fluor 546, Alexa Fluor 555, Alexa Fluor 568, Alexa Fluor 594, Alexa Fluor 610, Alexa Fluor 633, Alexa Fluor 647, Alexa Fluor 660, Alexa Fluor 680, Alexa Fluor 700, and, Alexa Fluor 750; amine-reactive BODIPY dyes, such as BODIPY 493/503, BODIPY 530/550. BODIPY 558/568. BODIPY 564/570. BODIPY 576/589. BODIPY 581/591. BODIPY 630/650, BODIPY 650/655, BODIPY FL, BODIPY R6G, BODIPY TMR, and, BODIPY-TR; Cy3, Cy5, 6-FAM, Fluorescein Isothiocyanate, HEX, 6-JOE, Oregon Green 488, Oregon Green 500, Oregon Green 514, Pacific Blue, REG, Rhodamine Green. Rhodamine Red. Renographin, ROX, SYPRO, TAMRA. 2’, 4’,5’,7^:-Tetrabromosulfonefluorescein, and TET.

[0121] Examples of dye/quencher pairs (i.e., donor/acceptor pairs) include, but are not limited to, fluorescein/tetramethylrhodamine; lAEDANS/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, FRET may be detected, in some embodiments, by fluorescence depolarization. Certain specific examples of dye/quencher pairs (i.e., donor/acceptor pairs) include, but are not limited to, Alexa Fluor 350/ Alexa Fluor488; Alexa Fluor 488/ Alexa Fluor 546; Alexa Fluor 488/ Alexa Fluor 555; Alexa Fluor 488/Alexa Fluor 568; Alexa Fluor 488/ Alexa Fluor 594; Alexa Fluor 488/Alexa Fluor 647; Alexa Fluor 546/ Alexa Fluor 568; Alexa Fluor 546/ Alexa Fluor 594; Alexa Fluor 546/ Alexa Fluor 647; Alexa Fluor 555/Alexa Fluor 594; Alexa Fluor 555/Alexa Fluor 647; Alexa Fluor 568/ Alexa Fluor 647; Alexa Fluor 594/ Alexa Fluor 647; Alexa Fluor 350/QSY35; Alexa Fluor 350/dabcyl; Alexa Fluor 488/QSY 35; Alexa Fluor 488/dabcyl; Alexa Fluor 488/QSY 7 or QSY 9; Alexa Fluor 555/QSY 7 or QSY9; Alexa Fluor 568/QSY 7 or QSY 9; Alexa Fluor 568/QSY 21; Alexa Fluor 594/QSY 21; and Alexa Fluor 647/QSY 21. In some instances, the same quencher may be used for multiple dyes, for example, a broad spectrum quencher, such as an Iowa Black® quencher (Integrated DNA Technologies, Coralville, IA) or a Black Hole Quencher™ (BHQ™; Sigma-Aldrich, St. Louis, MO).

[0122] Specific examples of fluorescently labeled ribonucleotides useful in the preparation of probes for use in some embodiments of the methods described herein are available from Molecular Probes (Invitrogen), and these include, Alexa Fluor 488-5- 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.

[0123] Specific examples of fluorescently labeled deoxyribonucleotides useful in the preparation of probes for use in the methods described herein include Dinitrophenyl (DNP)-l'-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, 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. Fluorescently labeled nucleotides are commercially available and can be purchased from, e.g.. Invitrogen.

[0124] As noted above, exemplary detectable moieties also include members of binding pairs. Exemplary binding pairs include, but are not limited to, biotin and streptavidin, antibodies and antigens, etc.

[0125] In some examples, the sample is selected from a sputum sample, a nasal aspirate sample, a nasal wash sample, a nasal swab sample, a nasopharyngeal swab sample, a saliva sample, an oropharyngeal swab sample, a throat swab sample, a bronchoalveolar lavage sample, a bronchial aspirate sample, a bronchial wash sample, an endotracheal aspirate sample, an endotracheal wash sample, a tracheal aspirate sample, a nasal secretion sample, a mucus sample, a pleural effusion sample, a cerebrospinal fluid sample, a stool sample, a tissue biopsy sample, a breath sample, or a combination thereof.

Sample

[0126] The sample to be tested can be any sample suspected of containing at least one target region, for example, anon-tuberculosis mycobacterium (NTM) or a respiratory pathogen biomarker. In some embodiments, the sample is a biological sample collected from a subject. In other embodiments, the sample is a sample that is not collected directly from a subject, such as, e.g., a wastewater sample or a sample from an air filter in a building.

[0127] Illustrative biological samples include samples of bodily fluids, such as nasal aspirates, nasal washes, nasal swabs, nasopharyngeal swabs, saliva, oropharyngeal swabs, throat swabs, bronchoalveolar lavage samples, bronchial aspirates, bronchial washes, endotracheal aspirates, endotracheal washes, tracheal aspirates, nasal secretion samples, mucus samples, sputum samples, lung tissue samples, etc.

[0128] The sample to be tested is. in some embodiments, fresh (i.e., never frozen). In other embodiments, the sample is a frozen specimen. 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. [0129] In some embodiments, a sample to be tested is contacted with a buffer after collection. For example, in the case of a nasal aspirate sample or nasal wash sample or a sample derived from a nasal aspirate sample or nasal wash sample, a buffer (including, e g., a preservative) can be added to the nasal aspirate sample or nasal wash sample. In embodiments where the sample is a nasopharyngeal swab sample, the swab can simply be placed in a buffer. In some embodiments, that sample is contacted with the buffer immediately; in the case of a swab, the swab is immediately placed in the buffer. In some embodiments, the sample (e.g., including the swab) is contacted with buffer within about 5 minutes, within about 10 minutes, within about 30 minutes, within about 1 hour, or within about 2 hours of sample collection.

[0130] In some embodiments, 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 of sample or buffered sample are used in the present methods. In some embodiments, from about 0. 1 ml to about 1 ml of sample or buffered sample is used in the present methods. Subjects

[0131] A biological sample useful in the methods described herein can be collected from any subject that can be infected by one, several, or all of the respiratory pathogens described above. In various embodiments, the subject can include nonhuman animals, e.g., canines, felines, equines, primates, and other non-human mammals, as well as humans.

[0132] In some embodiments, the sample to be tested is obtained from an individual who has one or more symptoms of tuberculosis or influenza infection. In some embodiments, the sample to be tested is obtained from an individual who has previously been diagnosed with a condition caused by a respiratory pathogen (e.g., influenza or Covid-19). In some such embodiments, the individual is monitored for recurrence of a condition caused by a respiratory pathogen (e.g., influenza or Covid- 19).

[0133] In some embodiments, methods described herein can be used for routine screening of apparently healthy individuals with no risk factors. In some embodiments, methods described herein are used to screen asymptomatic individuals, for example, during routine or preventative care. In some embodiments, methods described herein are used to screen women who are pregnant or who are attempting to become pregnant. [0134] In some embodiments, the methods described herein can be used to assess the effectiveness of a treatment in an individual undergoing treatment a condition caused by a respiratory pathogen (e g., influenza or Covid- 19).

Assay Methods

[0135] The methods for detecting melt profile (e.g., melt temperature) signatures for at least two target regions of a nucleic acid in a multiplex amplification reaction, as disclosed herein can be more specifically described as comprising: a) contacting each target region with a set of primer and a set of two or more probes, wherein each of the two or more probes in the set for the target region comprises a detectable label that emits light at the same wavelength; b) subjecting the target regions, primers, and probes to amplification conditions to amplify the target regions; and c) generating and detecting a melt temperature signature specific for each target region, wherein the melt temperature signature comprises a plurality of melt profiles generated from the set of two or more probes hybridizing to the amplified target region. [0136] The methods can utilize RNA targets for detection by direct hybridization or. more easily, by reverse transcription of a target RNA to produce a cDNA that is complementary to the target RNA. This cDNA can be directly detected by direct hybridization or by amplification of the cDNA template.

[0137] Nucleic acid amplification provides rapid, sensitive, and specific detection of nucleic acid targets, and has been employed in a wide variety of assay formats to detect nucleic acid targets. Those of skill in the art can, following the guidance herein, carry out the methods described herein in any number of different nucleic acid amplification-based assays, using, for example, any of the nucleic acid amplification methods discussed above. Such methods can entail thermocy cling, but need not do so, 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 to a DNA, such as provided by the SenseAmp Plus™ Kit available at Implen, Germany, or temperature oscillation. Amplification and detection can be earned out in solution or can make use of a solid support (e.g., a biochip). Nucleic acid amplification-based assays can employ a single reaction chamber or multiple reaction chambers. Amplification can be nested or non-nested. In some embodiments, detection includes electrochemical detection.

[0138] In some embodiments of amplification by polymerase chain reaction (PCR). an exemplary’ cycle comprises an initial denaturation at from about 90°C to about 100°C for about 20 seconds to about 5 minutes, followed by cycling that comprises denaturation at from about 90°C to about 100°C for about 1 to about 10 seconds, followed by annealing and amplification at from about 60°C to about 75°C for about 10 to about 40 seconds. A further exemplary cycle comprises about 20 seconds at about 94°C, followed by up to 3 cycles of 1 second at about 95°C, about 35 seconds at about 62°C, 20 cycles of 1 second at about 95°C, about 20 seconds at about 62°C, and 14 cycles of 1 second at about 95°C, about 35 seconds at about 62°C. In some embodiments, for the first cycle following the initial denaturation step, the cycle denaturation step is omitted. In some embodiments, Taq polymerase is used for amplification. In some embodiments, the cycle is carried out at least 10 times, at least 15 times, at least 20 times, at least 25 times, at least 30 times, at least 35 times, at least 40 times, or at least 45 times, and any value in between (e.g., at least 27 times). In some embodiments, Taq is used with a hot-start function. In some embodiments, detection of the target regions occurs in less than 3 hours, less than 2.5 hours, less than 2 hours, less than 1 hour, or less than 30 minutes (and any value in between, e.g., less than 2. 13 hours) from initial denaturation through the last extension. In some embodiments, target regions are detected by a method that includes real-time quantitative PCR, e.g., using FRET probes, such as those described above.

[0139] In some embodiments, amplification can be by non-isothermal amplification.

[0140] As described herein, the methods comprise generating unique melt profile for each target region of nucleic acid. The melt profile signature can include melt temperature (melt peak) signature, melt curve pattern (shape), or a combination thereof. In some instances, the methods comprise deriving at least one melt peak temperature (T_m), preferably at least two T_ms from the plurality of melt profiles for each target region, to detect and optionally identify each target region. In one embodiment, the methods comprise deriving at least two T_ms from the plurality of melt profiles for each target region, preferably wherein the T_ms are separated by at least 4°C. Accordingly, the methods can comprise differentiating and identifying each target region of nucleic acid detected.

[0141] Exemplary target regions of nucleic acid, as described herein, can include bacterial nucleic acids (e.g., Mycobacterium tuberculosis gene), viral nucleic acids, or a combination thereof. For example, the nucleic acid can include a Mycobacterium tuberculosis gene, preferably selected from an antimicrobial resistance Mycobacterium tuberculosis gene, an antimicrobial susceptible Mycobacterium tuberculosis gene, a nontuberculous mycobacterium, a wild-type Mycobacterium tuberculosis gene, or a combination thereof.

[0142] As described herein, a melting profile (e.g., melting point)-based detection method is used for detecting the presence of the target region of nucleic acids. For example, detection can be by obtaining melting curves for the amplified products (e.g., in a real-time PCR assay), by fluorophore-labelled nucleic acid probes, mass spectrometry or by sequencing of the amplified products. Amplification products will exhibit different melting curves depending on the type and number of nucleic acid variants in the amplification product. Methods for determining melting curves have been described and are known to those of skill in the art and any such methods for determining melting curves can be employed with the methods of the present disclosure. In a preferred embodiment of the methods disclosed herein, detection probes labeled with a detectable moiety (e.g., fluorophore group and a quencher group) can be used to carry out melting curve analysis. In brief, at an ambient temperature, the two or more detection probes and their complementary sequence can form a duplex by virtue of base pairing. In this case, the detectable moiety (e.g., a fluorescent group) and the quencher group on the probes are separated from each other, the quencher group cannot absorb the signal generated by the reporter group (e.g., a fluorescent signal), and the signal (e.g., a fluorescent signal) can be detected. With the increase of temperature, the two strands of the duplex begin to dissociate (i.e. the detection probes is gradually dissociated from its complementary sequence), and the dissociated detection probes are in a single-stranded and randomly coiled state. In this case, the fluorescent group and the quencher group on the dissociated detection probes are close to each other, and therefore the signal (e.g., a fluorescent signal) generated by the reporter group (e.g., a fluorescent group) is absorbed by the quencher group. Therefore, with the increase of temperature, the detected signal (e.g., a fluorescent signal) gradually weakens. When the two strands of the duplex are completely dissociated, the detection probes are in a single-stranded and randomly coiled state. In this case, the signals (e.g., fluorescent signals) generated by the fluorescent groups on the detection probes are absorbed by the quencher groups. Therefore, the signals (e.g., fluorescent signals) generated by the fluorescent groups cannot be detected substantively. Therefore, by detecting the signal (e g., a fluorescent signal) generated by a duplex comprising two or more detection probes during heating or cooling, the hybridization and dissociation process of detection probes and their complementary sequence can be observed, and a melt curve signature, in which the signal intensity’ changes with a change in temperature, is formed. Further, by derivation analysis of the obtained curve(s), a curve (i.e., the melting curve of the duplex), in which the rate of change in signal intensity⁷ is used as the ordinate and the temperature is used as the abscissa, can be obtained. The peak in each melting curve is the melting peak, and the temperature corresponding to the peak it is the melting point (T_m value) of said duplex. In general, the higher the matching degree between a detection probe and its complementary sequence is (e.g., fewer bases are mismatched, and more bases are paired), the higher the T_m value of a duplex is. Therefore, by detecting the T_m value of a duplex, the presence and identity of the sequence complementary to the detection probe in the duplex can be determined. As used herein, the term “melting temperature’", “melting peak”, “melting point"’ and “T_m value” have the same meaning and can be used interchangeably.

[0143] In some embodiments, quantitation of the results of real-time PCR assays is done by constructing a standard curve from a nucleic acid of known concentration and then extrapolating quantitative information for target regions of unknown concentration. In some embodiments, the nucleic acid used for generating a standard curve is a DNA (for example, an endogenous control, or an exogenous control). In some embodiments, the nucleic acid used for generating a standard curve is a purified double-stranded plasmid DNA or a single-stranded DNA generated in vitro. The methods disclosed herein can employ a primer pair that selectively hybridizes to the exogenous control and/or the endogenous control. The exogenous control is a sample processing control, and wherein the endogenous control is a sample adequacy control.

[0144] In some embodiments, in order for an assay to indicate that a given target region is not present in a sample, the Ct values for an endogenous control (such as an SAC) and/or an exogenous control (such as an SPC) must be within previously- determined valid ranges. For example, in some embodiments, the absence of a particular target region cannot be confirmed unless the controls are detected, indicating that the assay was successful.

[0145] In some embodiments, a threshold Ct (or a “cutoff Ct”) value for a target region (including an endogenous control and/or exogenous control), below which the gene is considered to be detected, has previously been determined. In some embodiments, a threshold Ct is determined using substantially the same assay conditions and system (such as a GeneXpert®) on which the samples will be tested. [0146] Real-time PCR is performed using any PCR instrumentation available in the art. Typically, instrumentation used in real-time PCR data collection and analysis comprises a thermal cycler, optics for fluorescence excitation and emission collection, and optionally a computer and data acquisition and analysis software.

[0147] In some embodiments, the number of target regions in an assay exceeds the number of labels that can be detected, e.g., in particular instruments. Therefore, the PCR amplification can include both Ct and melt analysis to increase the number of possible reported results. [0148] Another approach to detect target regions can include high-resolution melt. Target regions can also be detected by real-time PCR but in more than one reaction chambers. Examples of other approaches that can be employed in the methods describe herein include bead-based flow cytometric assay. See Lu J., et al., (2005), Nature, 435:834-838, which is incorporated herein by reference for this description. In some embodiments, the approach for detecting a target region does not include beadbased flow cytometric assay, microfluidic devices and single-molecule detection, simple gel electrophoresis, use of a capture probe attached to a solid-support, separation of reaction mixture into multiple reaction chambers, array-based detection, nested amplification, electrochemical detection, high resolution melt only, or a combination thereof.

Automated Assay Methods

[0149] Readily automated approaches are of great interest in performing the methods for detecting melt profile (melt temperature) signatures for the target regions of nucleic acid. The methods can be carried out in a semi-automated or substantially automated manner using a commercially available nucleic acid amplification system. Exemplary nonlimiting nucleic acid amplification systems that can be used to carry out the methods include the GENEXPERT® system, a GENEXPERT® Infinity system, and GENEXPERT® Xpress System (Cepheid, Sunnyvale, Calif). In some embodiments, the amplification system may be available at the same location as the individual to be tested, such as a health care provider’s office, a clinic, or a communityhospital, so processing is not delayed by transporting the sample to another facility. Assays according to the method described herein can be completed in under 3 hours, in some embodiments, under 2 hours, in some embodiments, under 1 hour, using an automated system, for example, the GENEXPERT® system. The GENEXPERT® utilizes a self-contained, single-use cartridge. Sample extraction, amplification, and detection may all carried out within this self-contained sample cartridge as further described herein.

[0150] In certain embodiments, the method comprises: placing the nucleic acid sample in a sample chamber of a cartridge; and if the sample comprises cells, lysing the cells in the sample with one or more lysis reagents present within at least one of the plurality of chambers or capturing the cells in a filter within the cartridge and lysing the cells by means of ultrasonication to release sample nucleic acids; or if the sample comprises cell-free nucleic acids, capturing the free nucleic acids in a nucleic acid capture chamber and eluting the captured nucleic acid after washing to remove impurities.

[0151] After the sample is added to the cartridge, the sample is contacted with lysis buffer and released nucleic acid (NA) is bound to aNA-binding substrate, such as a silica or glass substrate. The sample supernatant is then removed and the NA eluted in an elution buffer, such as a Tris/EDTA buffer. The eluate may then be processed in the cartridge to detect target regions as described herein. In some embodiments, the eluate is used to reconstitute at least some of the PCR reagents, which are present in the cartridge as lyophilized particles.

[0152] A cartridge having a plurality of chambers can have the set of primers and probes described herein, or a subset thereof, disposed in a chamber. In some embodiments, the set of primers and probes described herein, or a subset thereof, are disposed in more than one of the plurality of chambers.

[0153] In some embodiments, RT-PCR is used to amplify and analyze the presence of the target regions. In some embodiments, the reverse transcription uses MMLV and/or CAT-A RT enzyme and an incubation of from about 5 to about 20 minutes at about 40°C to about 50°C. In some embodiments, the PCR uses Taq polymerase with hot-start function, such as AptaTaq (Roche). In some embodiments, the initial denaturation is at about 90°C to about 100°C for about 20 seconds to about 5 minutes; the cycling denaturation temperature is from about 90°C to about 100°C for about 1 to about 10 seconds; the cycling anneal and amplification temperature is from about 60°C to about 75°C for about 10 to about 40 seconds; and up to 50 cycles are performed.

[0154] In some embodiments, a double-denature method is used to amplify low-copy number target regions. A double-denature method comprises, in some embodiments, a first denaturation step followed by addition of primers and/or probes for detecting target regions. All or a substantial portion of the nucleic acid-containing sample (such as a DNA eluate) is then denatured a second time before, in some instances, a portion of the sample is aliquoted for cycling and detection of the target regions. While not intending to be bound by any particular theory, the double-denature protocol may increase the chances that a low-copy number target region (or its complement) will be present in the aliquot selected for cycling and detection because the second denaturation effectively doubles the number of target regions (i.e., it separates the target region and its complement into two separate templates) before an aliquot is selected for cycling. In some embodiments, the first denaturation step comprises heating to a temperature of from about 90°C to about 100°C for a total time of about 30 seconds to about 5 minutes. In some embodiments, the second denaturation step comprises heating to a temperature of from about 90°C to about 100°C for a total time of about 5 seconds to about 3 minutes. In some embodiments, the first denaturation step and/or the second denaturation step is carried out by heating aliquots of the sample separately. In some embodiments, each aliquot may be heated for the times listed above. As a non-limiting example, a first denaturation step for an NA- containing sample (such as a DNA eluate) may comprise heating at least one, at least two, at least three, or at least four aliquots of the sample separately (either sequentially or simultaneously) to a temperature of from about 90°C to about 100°C for about 60 seconds each. As a non-limiting example, a second denaturation step for aNA- containing sample (such as a DNA eluate) containing enzyme, primers, and probes may comprise heating at least one, at least two, at least three, or at least four aliquots of the eluate separately (either sequentially or simultaneously) to a temperature of from about 90°C to about 100°C for about 5 seconds each. In some embodiments, an aliquot is the entire NA-containing sample (such as a DNA eluate). In some embodiments, an aliquot is less than the entire NA-containing sample (such as a DNA eluate).

[0155] In some embodiments, an off-line centrifugation is used, for example, with samples with low cellular content. The sample, with or without a buffer added, is centrifuged and the supernatant removed. The pellet is then resuspended in a smaller volume of either supernatant or the buffer. The resuspended pellet is then analyzed as described herein.

[0156] The assay methods described herein can be a point-of-care method. In some examples, the method can comprise detecting and differentiating the target regions in a sample within 150 minutes, within 140 minutes, within 130 minutes, or within 120 minutes of collecting the sample from the subject.

Exemplary Automation and Systems

[0157] Many existing fully integrated nucleic acid amplification and test systems capable of sample preparation are normally quite complicated and costly. The nucleic acid amplification and test systems provided herein perform rapid, simple, convenient, and affordable nucleic acid analysis.

System Overview

[0158] In one aspect, the disclosure pertains to a sample cartridge that utilizes a valve body platform that allows for detection of enveloped and free nucleic acids. In some embodiments, the valve body includes a sample processing region or lysing chamber that provides for either or both mechanical and chemical lysis. This allows a single cartridge to provide lysing for a multitude of differing types of targets. In some embodiments, the sample cartridge can perform processing and detection of targets requiring mechanical lysing and chemical lysing.

[0159] The sample cartridge device can be any device configured to perform one or more process steps relating to preparation and/or analysis of a biological fluid sample 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 further be configured to perform additional processes, such as detection of a target region in a nucleic acid amplification test (NAAT), e.g.. Polymerase Chain Reaction (PCR) assay, by use of a reaction vessel attached to the sample cartridge. In some embodiments, the reaction vessel extends from the body of the cartridge. Preparation of a fluid sample generally involves a series of processing steps, which can include chemical, electrical, mechanical, thermal, optical or acoustical processing steps according to a specific protocol. Such steps can be used to perform various sample preparation functions, such as cell capture, cell lysis, binding of analyte, and binding of unwanted material.

[0160] A sample cartridge suitable for use with the composition and methods disclosed herein, includes one or more transfer ports through which the prepared fluid sample can be transported into an attached reaction vessel for analysis. FIG. 3A illustrates an exemplary⁷ assay cartridge 100 suitable for sample preparation and analytics testing by PCR when received in an instrument module in accordance with some embodiments. The sample cartridge is attached with a reaction vessel 116 (also referred to as a “reaction tube” or “PCR tube”) adapted for analysis of a fluid sample processed within the sample cartridge 100. In some embodiments the reaction vessel extends from the cartridge body. Such a sample cartridge 100 includes various components including a main housing 102 having one or more chambers 108 for processing of the fluid sample, which typically include sample preparation before analysis. In these embodiments, the sample cartridge can be a fully integrated nucleic acid amplification and test system combining sample preparation, amplification, and detection together. The instrument module facilitates the processing steps needed to perform sample preparation and the prepared sample is transported through one of a pair of transfer ports into fluid conduit of the reaction vessel 116 attached to the housing of the sample cartridge 100. The prepared biological fluid sample is then transported into a reaction chamber of the reaction vessel where the biological fluid sample undergoes nucleic acid amplification. In some embodiments, the amplification is a polymerase chain reaction. In some embodiments, concurrent with the amplification of the biological fluid sample, an excitation means, and an optical detection means of the module is used to detect optical emissions that indicate the presence or absence of a target region of interest. It is appreciated that such a reaction vessel could include various differing chambers, conduits, or micro-well arrays for use in detecting the target analyte. The sample cartridge can be provided with means to perform preparation of the biological fluid sample before transport into the reaction vessel. Any chemical reagent required for cell lysis or means for binding or detecting an analyte of interest (e.g., reagent beads) can be contained within one or more chambers of the sample cartridge, and as such can be used for sample preparation. [0161] An exemplary use of a reaction vessel for analyzing a biological fluid sample is described in commonly assigned U.S. Patent No. 6,818,185, entitled “Cartridge for Conducting a Chemical Reaction,” filed May 30, 2000, the entire contents of which are incorporated herein by reference for all purposes. Examples of the sample cartridge and associated modules are shown and described in U.S. Patent No. 6,374,684, entitled “Fluid Control and Processing System” filed August 25, 2000, and U.S. Patent No, 8,048,386, entitled “Fluid Processing and Control,” filed February 25, 2002, U.S. Provisional Application No. 63/217,672 entitled “Universal Assay Cartridge and Methods of Use” filed July 1, 2021; U.S. Provisional Application No. 63/319,993 entitled “Unitary Cartridge Body and Associated Components and Methods of Manufacture” filed March 15, 2022; and U.S. Patent No. 10,562,030 entitled “Molecular Diagnostic Assay System” filed July 22, 2016; the entire contents of which are incorporated herein by reference in their entirety for all purposes. [0162] Various aspects of the sample cartridge 100 can be further understood by referring to U.S. Patent No. 6,374,684 “the ‘684 patent”), which described certain aspects of a sample cartridge in greater detail. Such sample cartridges can include a fluid control mechanism, such as a rotary fluid control valve assembly, that is fluidically connected to the chambers of the sample cartridge. The term “chamber” can be used interchangeably with the terms “well”, “tube”, and the like. Rotation of the rotary fluid control valve permits fluidic communication between chambers and the valve so as to control flow of a biological fluid sample deposited in the cartridge into different chambers in which various reagents can be provided according to a particular protocol as needed to prepare the biological fluid sample for analysis. To operate the rotary valve, the cartridge processing module comprises a motor such as a stepper motor that is typically coupled to a drive train that engages with a feature of the valve in the sample cartridge to control movement of the valve in coordination with movement of the syringe, thereby resulting movement of the fluid sample according to the desired sample preparation protocol. The fluid metering and distribution function of the rotary valve according to a particular sample preparation protocol is demonstrated in the ‘684 patent.

Exemplary Assay Cartridge and Valve Assemblies Overview

[0163] As shown in FIG. 3 A, the test cartridge 100 comprises a cartridge body 102 containing a plurality of chambers 108 for reagents or buffers and sample processing. The chambers are disposed around a central syringe barrel 106 that is in fluid communication with a valve body 110 (see FIGS. 3B and 3C) and that is sealed with a gasket 104. The valve body 110 can include a cap 112 and the entire cartridge body can be supported on a cartridge base 101. The valve body typically contains one or more channels or cavities (chamber(s) 114) that can contain a filter as described herein that can function to bind and elute a nucleic acid. In some embodiments the cartridge further comprises one or more temperature-controlled channels or chambers that can, in certain embodiments, function as thermocycling chambers. A “plunger” not shown can be operated to draw fluid into the syringe barrel 106 and rotation of the valve body 110 provides selective fluid communication between the various reagent chambers 108 and channels, reaction chamber(s), mixing chambers, and optionally, any temperature-controlled regions. Thus, the various reagent chambers 108, reaction chambers, filter material(s), and temperature-controlled chambers or channels are selectively in fluid communication 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, the various reagent chambers, reaction chambers, filter material, and temperature-controlled chambers or channels are selectively in fluid communication by linear progression (e g., by forced movement) of the reagents and sample from one chamber to the next.

Reaction Modules

[0164] In certain embodiments the cartridge is configured for insertion into a reaction module. The module is configured to receive the cartridge therein. In certain embodiments the reaction module provides heating plates to heat the temperature- controlled chamber or channel. The module can optionally additionally include a fan to provide cooling where the temperature-controlled channel or chamber is a thermocycling channel or chamber. Electronic circuitry can be provided to pass information (e.g., optical information) to a computer for analysis. In certain embodiments the module can contain optical blocks to provide 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, e.g., signal DNAs amplified for various PCR targets. In various embodiments an electrical connector can be provided for interfacing the module with a system e.g. system controller or with a discrete analy sis/ controller unit. In certain embodiments, the module also contains a controller that operates a plunger in the syringe barrel and the rotation of the valve body.

Analytical System

[0165] In certain embodiments a system (e.g.. a processing unit) is provided. The system includes an enclosure that is configured to support and power multiple sample processing modules, where each processing module is configured to hold and operate a removable cartridge. In some embodiments, the system is configured to operate the sample processing modules to perform a PCR assay for one or more target region analytes and optionally to determine the level of one or more target RNA/DNA sequences within a corresponding removable sample cartridge. Typically, the processing on a sample within the corresponding removable sample cartridge involves operating the cartridge to perform a method as described herein. In certain embodiments the system is configured to contain one sample processing module. In certain embodiments the system is configured to contain at least two or more sample processing modules (e.g., at least 4, 8. 12. 16, 20, 24, 28, 32, 64, 128 or more) sample processing modules. In some embodiments, the system provides a user interface that allows the user input operational instructions and/or to monitor operation of the cartridges to determine the presence and/or quantity of one or more nucleic acids. [0166] While the methods described herein are described primarily with reference to the GENEXPERT® cartridge by Cepheid Inc. (Sunnyvale, Calif.) (see, e g., FIG. 3A), it will be recognized, that in view of the teachings provided herein the methods can be implemented on other cartridge/microfluidic systems, including alternative cartridge designs having valve assemblies that involve multiple interfacing components, as well as cartridge body defined by multiple interfacing components to form the multiple chambers of the cartridges, for example, those described in Korean Application No. 102293717B1 and KR102362853B1, cartridges that utilizes ultrasonic waves to lyse cells in a biological sample, for example, those described in International Application No. WO2021/245390A1, cartridges and systems that utilizes an electrowetting grid for microdroplet manipulation and electrosensor arrays configured to detect analytes of interest, for example, those described in International Application No. WO2016/077341A2, cartridges that facilitate movement of nucleic acid from one chamber to the next chamber by opening a vent pocket, for example, those described in International Application No. WO2012/145730A2, multiplexed assay systems comprising a plurality of thermocycling units such that individual chambers can be heated, cooled, and/or compressed to mix fluid within the chamber or to propel fluid in the chamber into another chamber, for example, those described in International Application No. WO2015/138343A1, and as well as systems for rapid amplification of nucleic acids facilitated by flexible portions of the sample cartridge aligned to accomplish temperature cycling for nucleic acid amplification, for example, those described in International Application No.WO2017/147085Al. Such cartridge/microfluidic systems can include, for example microfluidic systems implemented using soft lithography, rmcro/nano-fabricated microfluidic systems implemented using hard lithography, and the like.

[0167] In certain embodiments, the cartridge for detecting a melt profile signature for target regions of a nucleic acid in a multiplex amplification reaction comprises a) a cartridge body comprising a plurality’ of chambers therein, wherein the plurality of chambers includes: a sample chamber having at least a fluid outlet in fluid communication with another chamber of the plurality; and an optional lysis chamber in fluidic communication with the sample chamber, optionally wherein the sample chamber and lysis chamber are the same; b) a reaction vessel fluidically coupled to the plurality of chambers of the cartridge body and configured for: i) amplification of nucleic acid and ii) detection and identification of a plurality of amplification products via real-time PCR; c) a filter disposed in a fluidic path between the lysis chamber, if present, or the sample chamber, and the reaction vessel, and d) a set of primer and a set of two or more probes for each of the at least two target regions, wherein each of the two or more probes in the set for a target region comprises a detectable label that emits light at the same wavelength.

[0168] The reaction vessel comprises one or more reaction chambers for amplification and detection of the amplification products. In certain embodiments, the reaction vessel comprises one reaction chamber for amplification and detection of the amplification products. Accordingly, the nucleic acid, primers, and probes are present in a single reaction solution, and wherein generating and detecting the melt temperature signatures for the target regions are from the single reaction solution. In certain embodiments, the reaction vessel comprises more than one reaction chambers for amplification and detection of the amplification products, for example, two reaction chambers, three reaction chambers, or four reaction chambers. Each reaction chamber can be configured to detect a single amplification product or a plurality of amplification products.

[0169] The cartridge can be a Clinical Laboratory Improvement Amendments (CLIA)-compliant cartridge. Additionally, it is appreciated that the assay methods described herein can further be realized in entirely different systems, including: isothermal nucleic acid amplification systems, digital RT-PCR, electrochemical PCR, lateral flow testing cartridges, electrochemical sensors, nucleic acid sequencing, CRISPR/Cas based technologies, chemiluminescence, and nanoparticle-based colorimetric detection.

[0170] In various embodiments, the signal DNA(s) from PCR (nucleic acid amplification) reactions are amplified for detection and quantification. In certain embodiments, the amplification comprise any of a number of methods including, but not limited to polymerase chain reaction (PCR). ligase chain reaction (LCR). ligase detection reaction (LDR), multiplex ligation-dependent probe amplification (MLP A), ligation followed by Q-replicase 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), and the like.

[0171] In illustrative, but non-limiting embodiments, the amplification reaction may produce an optical signal that is proportional to the amount of amplified target region (e.g., signal DNA). Illustrative optical signals include, but are not limited to a fluorescent signal, a chemiluminescent signal, an electrochemiluminescent signal, a colorimetric signal, and the like. In certain embodiments the optical signal is a fluorescent optical signal generated by a fluorescent indicator. In certain embodiments the fluorescent indicator is a non-specific intercalating dye that binds to doublestranded DNA products, while in certain other embodiments, the fluorescent indicator comprises a target sequence-specific probe (e.g., a TAQMAN® probe, a SCORPION® probe, a MOLECULAR BEACON®, and the like).

[0172] Single PCR reactions (nucleic acid amplification), or multiple PCR reactions (nucleic acid amplifications) run sequentially (or simultaneously in separate temperature controlled channels or chambers) can also use the same detectable label since sequentially run PCR signal DNAs are analyzed sequentially and the simultaneous PCR signal DNAs are distinguished by the occurrence in different temperature controlled channels or chambers. The signal produced by this amplification can be distinguished from other amplification products because it is not run at the same time and/or because it is run in a different reaction channel/chamber. However, where multiple nucleic acid amplifications are run simultaneously in the same chamber the reaction products of for each analysis are typically detected and/or quantified by the use of unique melt profiles and/or different and distinguishable labels.

[0173] In certain embodiments, amplification products (amplified nucleic acid from nucleic acid analysis) can be detected using methods well known to those of skill in the art. In certain embodiments the amplification is a straightforward simple PCR amplification reaction. In certain embodiments, however, a nested PCR reaction is used to amplify the nucleic acid from the nucleic acid analysis. In various embodiments, multiplexed PCR assays are contemplated, particularly where it is desired to analyze multiple products of the nucleic acid analysis in the same amplification reaction. In certain embodiments in such multiplexed amplification reactions, probe(s) for each specific target region has its own specific dye/fluorophore so that it is detectable independently of the other probes. In certain embodiments, typically, for signal generation, the probes used in various amplification reactions utilize a change in the fluorescence of a fluorophore due to a change in its interaction with another molecule or moiety brought about by changing the distance between the fluorophore and the interacting molecule or moiety for detection and/or quantification of the amplified product. Alternatively, other methods of detecting a polynucleotide in a sample, including, but not limited to, the use of radioactively labeled probes, are contemplated.

Exemplary Assay Configurations

Reagents for Assay

[0174] As described herein, the nucleic acid can be bound to a nucleic acidbinding substrate, also referred to herein as a filter. In some examples, the filter comprises glass fibers and optionally a polymeric binder. The glass fibers may be modified with a nucleic acid binding ligand such as an alkylamine, a cycloalkylamine, an alkyloxy amine, a polyamine moiety, an arylamine, an intercalating agent, a DNA groove binder, a peptide, an amino acid, a protein, or a combination thereof. In some examples, the filter comprises a 500 micron to 2000 microns thick glass fiber disk having a pore size of 0.2 microns to 1 micron. In some aspects, the sample can be contacted with a binding reagent, wash reagent, or a combination during or after lysis. The binding reagent can promote binding of nucleic acids to the filter, facilitating the removal of non-target material. In some embodiments, the binding reagent can include a binding polymer such as polyacrylic acid (PAA), polyacry lamide (PAM), polyethylene glycol (PEG), poly(sulfobetaine), or a salt, or combinations thereof. In some embodiments, the filtering reagent and/or the washing reagent can include the binding reagent. For example, the binding reagent, the filtering reagent, and/or the washing reagent can include a binding polymer (e.g., PEG 200), buffer, inorganic salt(s), antioxidant and/or chelating agent, antifoam SE15, sodium azide, disaccharide or disaccharide derivative, carrier protein, a chaotropic agent (such as guanidium hydrochloride) detergent, DMSO, or a combination thereof. The binding polymer can be present in an amount 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 or can fall within any range bounded by any of these values, e.g., from 10% to 60% v/v, of the binding reagent, filtering reagent, and/or the washing reagent. The buffer can be selected from the group consisting of Tris, 2-amino-2-hydroxymethyl- 1,3-propanediol, HEPES, phosphate buffer, PBS, citrate buffer, TAPS, Bicine, Tricine, TAPSO, HEPES, TES, MOPS, PIPES, Cacodylate, SSC, and MES. The concentration of the buffer can range from about 5 mM to about 100 mM, such as from about 5 rnM to about 50 mM. The salt, such as NaCl, KC1, or MgCh, can be present at a concentration from about 0.05 M to about 1 M, such as from about 0. 1 M to about 0.5 M. The antioxidant and/or chelating agent comprises an agent selected from the group consisting of N-acetyl-L-cysteine, ethylenediaminetetraacetic acid (EDTA), diethylene triamine pentaacetic acid (DTP A). ethylenediamine-N,N'-disuccinic acid (EDDS), 1,2- bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA), and a phosphonate chelating agent. In some embodiments the antioxidant and/or chelating agent comprises EDTA. In certain embodiments the antioxidant and/or chelating agent comprise 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 reagent, filtering reagent, and/or the washing reagent. In some embodiments the concentration of the antioxidant and/or chelating agent in the binding reagent, filtering reagent, or the washing reagent ranges from about 2 mM to about 50 mM or about 5 rnM to about 20 mM. In some embodiments, the detergent is an ionic detergent or a non-ionic detergent. The detergent can be selected from an ionic detergent or a non-ionic detergent. In some examples, the detergent comprises a detergent selected from the group consisting of N-lauroylsarcosine, sodium dodecyl sulfate (SDS), cetyl methyl ammonium bromide (CTAB), TRITON®-X-100, n-octyl-P-D-glucopyranoside, CHAPS, n-octanoylsucrose, n-octyl-P-D-maltopyranoside, n-octyl-P-D- thioglucopyranoside, PLURONIC® F-127, TWEEN® 20, Brij-35, and n-hepl l-p-D- glucopyranoside. The detergent can comprise about 0.1% to about 2% of the binding reagent, filtering reagent, and/or the washing reagent, and/or ranges from about 10 rnM up to about 100 mM. The binding reagent, filtering reagent and/or the washing reagent can have a pH ranging from about pH 6.0 to about pH 8.0 (such as from about 6.5 to about 7.5).

[0175] The sample supernatant is then removed and the nucleic acid is eluted in an elution buffer such as a Tris/EDTA buffer. The elution buffer can comprise ammonia or an alkali metal hydroxide. In general, the elution buffer has a pH above about 9, above about 10. or above about 11. The elution buffer can further comprise a poly anion, optionally a carrageenan, a carrier nucleic acid, or i-carrageenan and KOH. The eluate may then be processed in the cartridge to detect target genes as described herein. In some embodiments, the eluate is used to reconstitute at least some of the PCR reagents, which are present in the cartridge as lyophilized particles. Particularly, the lyophilized particles can be in the form of beads and comprise primers, probes, a salt, dNTPs, a thermostable polymerase, a reverse transcriptase, or a combination thereof. The lyophilized can be present in the reaction vessel of the cartridge.

[0176] As would be appreciated by the skilled artisan, a Ct value is the number of cycles in a quantitative PCR experiment that are required for the fluorescent signal associated with the amplification of a specific target region to exceed a predetermined threshold value. As would be appreciated by the skilled artisan, this threshold value can be the background fluorescence levels measured in the experiment.

[0177] The methods described herein can be carried out at the same facility’ where the biological sample was collected from a subject. For example, the method can be a point-of-care method. In other instances, the method can be carried out in a hospital, an urgent care center, an emergency room, a physician’s office, a health clinic, or a home. In further instances, the method is a Clinical Laboratory Improvement Amendments (CLIA)-waived test. In some embodiments, information concerning the diagnosis of HER2 expression level in the subject is communicated to a medical practitioner. A “medical practitioner,” as used herein, refers to an individual or entity that diagnoses and/or treats patients, such as a hospital, a clinic, a physician’s office, a physician, a nurse, or an agent of any of the aforementioned entities and individuals. In some embodiments, the methods are carried out at a laboratory that has received the subject’s sample from the medical practitioner or agent of the medical practitioner. The laboratory carries out the detection by any method, including those described herein, and then communicates the results to the medical practitioner. A result is “communicated,” as used herein, when it is provided by any means to the medical practitioner. In some embodiments, such communication may be oral or written, may be by telephone, in person, by e-mail, by mail or other courier, or may be made by directly depositing the information into, e g., a database accessible by the medical practitioner, including databases not controlled by the medical practitioner. In some embodiments the result of the assay is combined with clinical parameters, data, or information about other risk factors to make a diagnosis. In some embodiments, the information is maintained in electronic form. In some embodiments, the information can be stored in a memory or other computer readable medium, such as RAM, ROM, EEPROM, flash memory, computer chips, digital video discs (DVD), compact discs (CDs), hard disk drives (HDD), magnetic tape, etc. The results may also be provided using a web-based application that may be provided to the health care practitioner or to the patient on a smart phone or other mobile device. In some aspects, results may be provided to the patient via a mobile device.

[0178] In some embodiments, the method further comprises receiving a communication from the laboratory that indicates the diagnosis in the sample. A “laboratory,’' as used herein, is any facility that detects the target gene in a sample by any method, including the methods described herein, and communicates the result to a medical practitioner. In some embodiments, a laboratory is under the control of a medical practitioner. In some embodiments, a laboratory is not under the control of the medical practitioner.

Exemplary Detection Methods, Results, and Handling of Results

[0179] Before the start of the PCR reaction, the GENEXPERT® System measures the fluorescence signal from the probes to monitor bead rehydration, reaction tube fdling, probe integrity, and dye stability. This Probe Check Control (PCC) passes if it meets validated acceptance criteria.

[0180] In some embodiments, a computer-based analysis program is used to translate the raw data generated by the detection assay into data of predictive value for a clinician. The clinician can access the predictive data using any suitable means. Thus, in some embodiments, the present invention provides the further benefit that the clinician, who is not likely to be trained in genetics or molecular biology, need not understand the raw data. The data is presented directly to the clinician in its most useful form. The clinician is then able to immediately utilize the information in order to optimize the care of the subject.

[0181] When the GENEXPERT® System is used, the results are interpreted automatically and are shown in a “View Results” window.

[0182] The present disclosure contemplates any method capable of receiving, processing, and transmitting the information to and from laboratories conducting the assays, information provides, medical personal, and subjects. For example, in some embodiments of the present invention, a sample is obtained from a subject and submited to a testing service (e.g., clinical lab at a medical facility, genomic profiling business, etc.), located in any part of the world (e.g., in a country different than the country where the subject resides or where the information is ultimately used) to generate raw data. Where the sample comprises a tissue or other biological sample, the subject may visit a medical center to have the sample collected and sent to the testing service, or subjects may collect the sample themselves and directly send it to a testing service. Where the sample includes previously determined biological information, the information may be directly sent to the testing service by the subject (e.g., an information card containing the information may be scanned by a computer and the data transmited to a computer of the profiling center using an electronic communication systems). Once received by the testing service, the sample is processed and a set of test results is produced, specific for the diagnostic or prognostic information desired for the subject.

[0183] The test results can be prepared in a format suitable for interpretation by a treating clinician. For example, rather than providing raw expression data, the prepared format may represent a diagnosis or risk assessment for the subject, with or without recommendations for particular treatment options. The test results may be displayed to the clinician by any suitable method. For example, in some embodiments, the testing service generates a report that can be printed for the clinician (e g., at the point of care) or displayed to the clinician on a computer monitor.

[0184] In some embodiments, the 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 convert the raw data to information useful for a clinician or patient. The central processing facility provides the advantage of privacy (all data is stored in a central facility with uniform security protocols), speed, and uniformity of data analysis. The central processing facility can then control the fate of the data following treatment of the subject. For example, using an electronic communication system, the central facility can provide data to the clinician, the subject, or researchers. [0185] In some embodiments, the subject is able to directly access the data using the electronic communication system. The subject may choose further intervention or counseling based on the results. In some embodiments, the data is used for research use. For example, the data may be used to further optimize the inclusion or elimination of markers as useful indicators of a particular condition or stage of disease or as a companion diagnostic to determine a treatment course of action.

[0186] In some embodiments, the methods disclosed herein comprise administering a treatment regimen to the subject based on the determined target region present.

Kits

[0187] Also contemplated is a kit for carrying out the methods described herein. Such kits include one or more reagents useful for practicing any of these methods. In some embodiments, a kit includes primer pairs for amplifying and/or detecting the nucleic acid targets described herein, with probes specific for these targets. In some embodiments, these kits can include a set of primer and a set of two or more probes for each target region, wherein each of the two or more probes in the set for a target region comprises a detectable label that emits light at the same wavelength. The kit may further comprise one or more lysis reagents for releasing nucleic acid from the biological sample.

[0188] A kit generally includes a package with one or more containers holding the reagents, as one or more separate compositions or, optionally, as an admixture where the compatibility of the reagents will allow. The kit can also include other material(s) that may be desirable from a user standpoint, such as a buffer(s), a diluent(s), a standard(s). and/or any other material useful in sample processing, washing, or conducting any other step of the assay.

[0189] Kits preferably include instructions for carry ing out one or more of the screening methods described herein. Instructions included in kits can be affixed to packaging material or can be included as a package insert. While the instructions are typically written or printed materials they are not limited to such. Any medium capable of storing such instructions and communicating them to an end user can be employed. Such media include, but are not limited to, electronic storage media (e.g., magnetic discs, tapes, cartridges, chips), optical media (e.g., CD ROM), and the like. As used herein, the term ‘'instructions” can include the address of an internet site that provides the instructions.

[0190] In some embodiments, the kit can include any of the reagents described above provided with or in one or more GENEXPERT® cartridge(s). See e.g., US Patents 5,958,349, 6,403,037, 6,440,725, 6,783,736, 6,818, 185; each of which is herein incorporated by reference for this description).

[0191] In certain embodiments, the kit can optionally further include a sterile swab (e.g., an alcohol swab) for cleaning the sample site, and/or a drying pad (e.g., a gauze pad) for drying the site, and/or a dressing (e.g. bandage) for dressing the site after obtaining the sample.

[0192] In certain embodiments, the components for a single collection operation are packaged together in a packet. Such packets can include, for example, a single use disposable sample device, optionally a sterile swab, optionally a dr ing pad, and optionally a dressing. In certain embodiments the kit includes at least 2 packets, or at least 3 packets, or at least 4 packets, or at least 5 packets, or at least 6 packets, or at least 7 packets, or at least 8 packets.

[0193] In certain embodiments, the kit can further contain instructional materials teaching collection methods utilizing the kit components and. optionally, providing guidance to overcome problems that may occur during collection. The instructional materials can also include information and/or instructions regarding the use of the lysis reagent and/or instructions for the collection, and/or storage, and/or shipping of a cell or tissue sample. In certain embodiments the kits additionally contain reagents and/or instructions teaching the use of the lysis buffer for isolation and recovery of a nucleic acid.

[0194] Often and typically the instructional materials are provided in written form and can be printed on the kit components themselves (e.g. on the cover of a box, container, or on an envelope), or can be provided as an insert/instructional page or booklet. While the instructional materials typically comprise written or printed materials, they are not limited to such. Any medium capable of storing such instructions and communicating them to an end user is contemplated by this invention. Such media include, but are not limited to electronic storage media (e.g., magnetic discs, tapes, cartridges, chips), optical media (e.g., CD ROM), and the like. Such media may include addresses to internet sites that provide such instructional materials.

EXAMPLES

Example 1: Two staggered probe approach in one optical channel (equimolar concentrations of S2P12s & S2P13s) in multiplex.

[0195] This example shows use of staggered probes (see FIG. 1) approach to obtain unique melt temperature (T_m) values for a particular target region of interest. The method is particularly useful when limited by the number of available fluorophores for target detection. Performance is maximized by utilizing the same fluorophore quencher combinations for the probes. For the 2 probes mentioned in this example, a fluorescent dye labelled CF5 was used as the fluorophore and a 3^?-end quencher.

[0196] In the experiment, equimolar concentrations of two staggered probes (S2P12s- Staggered Probe 1 and S2P13s - Staggered Probe 2 as shown in Table 1 below) were used to determine if melt data for the targets with more mismatches with either of the two probes can be generated and its effect on the overall melt data. Two control mastermixes were also prepared. The effect of having two staggered probes with the same fluorophore in the same mastermix was determined.

[0197] Protocol: Probes having oligo sequences as shown in Table 2 were prepared. Thirty (30) prototype cartridges were built with reagent beads, buffers, and retaining balls. Fresh synthetic target stocks were also prepared using le4 cp/pL synthetic NTM as target. TE (10/0. 1) buffer was used as NTC and for preparing dilutions if needed. A mastermix with 10: 1 rev:fwd primer and having 99 pL/reaction; luL target for a final reaction vol of 100 pL w as prepared. Table 1 below show s the components of the mastermix. One tube with 247.5 pL for each synthetic target (TE buffer as negative control) was prepared: a. abscessus (1), b. asiaticum (2), c. chelonae (6), d. Gastri (10), and e. NTC. To each tube, 2.5 pL of synthetic target of respective NTM concentration/TE was added. 100 pL of mastermix was added for each condition to be tested. Two cartridge per condition was ran.

Table 1: PCR Mastermix [0198] Results. In this example, a staggered probe approach was tested in one channel (CF5). Both the probes used for this experiment are Sloppy Molecular Beacons used at equimolar concentration. As seen in Figures 2A to 2C, two distinct T_m values were reported. This can be useful in high-level multiplexing where many sequences need to be uniquely identified.

Example 2: Two probe approach in different optical channels (CF3 and CFS) using Staggered probes

[0199] In this experiment, a two-probe approach was tested using synthetic NTM targets.

[0200] Protocol: Probes having oligo sequences as shown in Table 2 were prepared. Twelve (12) prototype cartridges were built with reagent beads, buffers, and retaining balls. Fresh synthetic target stocks were also prepared using le4 cp/pL synthetic NTM as target. TE (10/0. 1) buffer was used as NTC and for preparing dilutions if needed. Two mastermix with 10: 1 rev:fwd primer and having 99 pL/reaction; luL target for a final reaction vol of 100 pL were prepared. Tables 3 and 4 below show the components of the mastermix. One tube per mastermix was prepared with 148.5 pL for each synthetic target: a. abscessus (1), b. avium paraTB (4), c. chelonae (6), d. fortuitum (9), e. intracellulare (15), f. kansasii (16), and g. NTC. To each tube, 1.5 pL of synthetic target of respective NTM concentration was added. 100 pL of mastermix was added to the cartridge for each condition to be tested. One cartridge per condition was ran.

Table 2: Oligonucleotide sequences

Table 3: PCR Mastermix

Table 4: PCR Mastermix

[0201] Results. In the example above, the staggered probe approach was tested in different optical channels. Both the probes used for this experiment are Sloppy Molecular Beacons used at equimolar concentration. As seen in Table 5 below, the T_m data remains consistent even after changing channels and two clear Tm values reported for most of the targets tested here. Empty cells mean no T_m value reported on GX; the absence of a Tm value can be the target’s own unique signature. Based on this data, the same concept has been demonstrated in 3 channels for Sloppy Molecular Beacons. The performances of other probes in the mastermix are not affected by this change.

[0202] As indicated in Table 5, in some instances, there may be overlapping melt peaks, such as for M. abscessus, M. avium, and M. fortuitum using the S4 probe in CF3 channel. In such instances, another probe within the same optical channel or different optical channel can be relied on to break the tie, i.e., differentiate these targets.

Table 5: Melt temperature signatures

Example 3: Wet lab data with staggered approach with two staggered probes in each of CF3 and CF6 channels

[0203] In this experiment, different probe concentrations with S2P 12.1 s and S2P13s, S3P4s-L and S3_PAs probes were tested with several organisms. Stacking two staggered probes to generate unique melt signatures for organisms including MTB was investigated. A mastermix was prepared with two stacked molecular beacon probes (CF3) and two stacked molecular beacon and linear probes (CF6), having oligo sequence as shown in Table 6.

[0204] Protocol: Twenty (20) prototype cartridges were built. Fresh synthetic target stocks were also prepared using le4 cp/pL synthetic NTM as target. TE (10/0.1) buffer was used as NTC and for preparing dilutions if needed. A mastermix with 10: 1 rev:fwd primer and having 99 pL/reaclion: luL target for a final reaction vol of 100 pL was prepared. Table 7 below shows the components of the mastermix. One tube per mastermix with 99 pL for each synthetic target was set-up: a. abscessus (1), b. avium_paraTB (4), c. chelonae (6), d. fortuitum (9), e. intracellulare (15), f. kansasii (16), g. simiae (26), h. xenopi (36), i. MTB (33), and j. NTC. To each tube, 1 pL of synthetic target of respective NTM concentration was added. lOOpL of mastermix was added to the cartridge for each condition to be tested.

Table 6: Oligonucleotide Sequences

Table 7: PCR Mastermix

[0205] Results: In this example, two staggered molecular beacon probes were added together in CF3 and a staggered molecular beacon and a linear probe were added together in CF6 optical channels respectively. As seen from Table 8, two Tm values were reported for staggered probes with most of the organisms tested. The performance of the other probe (staggered probe 4 in this case) is not negatively impacted by the addition of the two staggered probes in CF3 and CF6. Empty cells mean no Tm value reported on GX.

Table 8: Melt temperature signature Example 4: Wet lab data with SMB and Linear probe approach

[0206] In this experiment, different probe concentrations, S3P4s-L and S3_PAs probes, were investigated against 9 organisms. An initial experiment with two staggered (Staggered Probe 3 - molecular beacon and Staggered Probe 5 - linear) probes in one mastermix was performed with both probes at equimolar (400 nM) concentration. Testing began with lower concentrations of the linear probe followed by the same higher concentrations of the SMB as results from the previous experiment suggested that linear probe might have partially/completely dampened the performance of SMB. Oligo sequences used are as shown in Table 9.

[0207] Protocol: Twenty (20) prototype cartridges were built. Fresh synthetic target stocks were also prepared using le4 cp/pL synthetic NTM as target. TE (10/0.1) buffer was used as NTC and for preparing dilutions if needed. A mastermix with 10:1 rev:fwd primer and having 99 pL/reaction: IpL target for a final reaction vol of 100 pL was prepared. Table 10 below shows the components of the mastermix. One tube per mastermix with 148.5 pL was set-up for each synthetic target: a. abscessus (1), b. avium_paraTB (4), c. chelonae (6), d. fortuitum (9), e. gordonae (12), f. intracellulare (15), g. kansasii (16), h. simiae (26), i. xenopi (36), and j. NTC. To each tube, 1.5 pL of synthetic target of respective NTM concentration and BG DNA/TE (10/0. 1) was added. IOOUL of mastermix w as added to two cartridges for each condition to be tested.

Table 9: Oligonucleotide Sequences

Table 10: PCR Mastermix

[0208] Results. In this example, two staggered probes were tested with the two- probe staggered approach. One Sloppy Molecular Beacon and a Linear probe were tested in the CF6 channel. Unlike the previous dataset, both the probes were added to the mastermix at different concentrations (lower cone, of linear and higher cone, of SMB). As seen from the Table 1 1, two distinct values for CF6 channel for most of the organisms tested here were observed. The performance of the other probes is not affected by the addition of two probes in the CF6 channel. Empty' cells mean no T_m value reported on GX.

Table 11 : Melt temperature signature

Claims

1. A method for detecting melt temperature signatures for at least two target regions of nucleic acid in a multiplex amplification reaction, the method comprising: a) contacting the nucleic acid with sets of primers and sets of two or more probes for detecting each target region, wherein each of the two or more probes in a set comprises a detectable label that emits light at the same wavelength; b) subjecting the nucleic acid, primers, and probes to amplification conditions to amplify the target regions; and c) generating and detecting a melt temperature signature specific for each target region present, wherein the melt temperature signature comprises a plurality of melt profiles generated from the set of two or more probes hybridizing to the amplified target region.

2. The method of claim 1, wherein the method comprises contacting the nucleic acid with sets of primer and sets of two or more probes for simultaneously detecting the at least 2 target regions, preferably at least 5 target regions, more preferably at least 10 target regions. w herein the detectable label used in the sets of two or more probes are the same.

3. The method of claim 1, wherein the method comprises contacting the nucleic acid with sets of primer and sets of two or more probes for simultaneously detecting the at least 2 target regions, preferably at least 5 target regions, more preferably at least 10 target regions, wherein the detectable label used in the sets of two or more probes are different.

4. The method of claim 1, wherein the method comprises contacting the nucleic acid with sets of primer and sets of tw o or more probes for simultaneously detecting at least 5 target regions, more preferably at least 10 target regions, wherein the detectable label used in a plurality of the sets of two or more probes are the same, and the detectable label used in a plurality of the sets of two or more probes are different.

5. The method of any one of claims 1-4, wherein each target region that hybridizes to the two or more probes in a set is at least forty (40) nucleotides in length.

6. The method of any one of claims 1-5, wherein the melt temperature signature for each target region comprises at least one melt peak temperature (T_m), preferably at least two T_ms.

7. The method of claim 6, wherein the melt temperature signature comprising two or more T_ms, the T_ms are separated by at least 4°C.

8. The method of any one of claims 1-7, wherein the sets of two or more probes comprises overlapping (staggered) hybridizing probes, sequential hybridizing probes, spaced out hybridizing probes, or a combination thereof.

9. The method of any one of claims 1-8, wherein the sets of two or more probes comprise a molecular beacon probe, a linear probe, a FRET (TaqMan) probe, or a combination thereof.

10. The method of any one of claims 1-9, wherein the sets of two or more probes comprise at least tw o sloppy molecular beacon probes, at least two linear probes, or a sloppy molecular beacon probe and a linear probe.

11. The method of any one of claims 1-10, wherein the detectable label comprises a fluorescent dye and a quencher molecule.

12. The method of any one of claims 1-11. wherein the method employs a primer pair that selectively hybridizes to an exogenous control and/or an endogenous control, wherein the exogenous control is a sample processing control, and wherein the endogenous control is a sample adequacy control.

13. The method of any one of claims 1-12. wherein the nucleic acid, primers, and probes are present in a single reaction solution, and wherein generating and detecting the melt temperature signatures for the target regions are from the single reaction solution.

14. The method of any one of claims 1-13, further comprising differentiating and identifying each target region.

15. The method of any one of claims 1-14, wherein the nucleic acid is selected from bacterial nucleic acid, viral nucleic acid, fungal nucleic acid, or a combination thereof.

16. The method of any one of claims 1-15, wherein the target regions include a Mycobacterium tuberculosis gene, a nontuberculous Mycobacterium gene, or a combination thereof.

17. The method of any one of claims 1-16, wherein the target regions include a nontuberculous Mycobacterium gene.

18. The method of claim 17, wherein the target regions include a nontuberculous Mycobacterium gene selected from Mycobacterium abscessus, Mycobacterium avium, Mycobacterium chelonae, Mycobacterium fortuitum, Mycobacterium gordonae, Mycobacterium intr acellular e, Mycobacterium kansasii, Mycobacterium simiae, Mycobacterium xenopi. or a combination thereof.

19. The method of any one of claims 1-18, wherein the sample is a sputum sample, a nasal aspirate sample, a nasal wash sample, a nasal swab sample, a nasopharyngeal swab sample, a saliva sample, an oropharyngeal swab sample, a throat swab sample, a bronchoalveolar lavage sample, a bronchial aspirate sample, a bronchial wash sample, an endotracheal aspirate sample, an endotracheal w ash sample, a tracheal aspirate sample, a nasal secretion sample, a mucus sample, a pleural effusion sample, a cerebrospinal fluid sample, a stool sample, a tissue biopsy sample, a breath sample, or a combination thereof.

20. The method of any one of claims 1-19, wherein the amplification comprises non-isothermal amplification, optionally by thermal cycling or temperature oscillation.

21. The method of any one of claims 1-20, wherein the method is performed via real-time PCR.

22. The method of any one of claims 1-21, wherein the method is a point-of- care method.

23. The method of any one of claims 1-22, wherein the method comprises detecting and differentiating the target regions in a sample within 150 minutes, within 140 minutes, within 130 minutes, or within 120 minutes of collecting the sample from the subject.

24. The method of any one of claims 1-23, wherein the method is a cartridge-based method and further comprises: placing the nucleic acid sample in a sample chamber of a cartridge; and if the sample comprises cells, lysing the cells in the sample with one or more lysis reagents present within at least one of the plurality of chambers or capturing the cells in a filter within the cartridge and lysing the cells by means of ultrasonication to release sample nucleic acid; or if the sample comprises cell-free nucleic acid, capturing the free nucleic acid in a nucleic acid capture chamber and eluting the captured nucleic acid after washing to remove impurities.

25. A cartridge for detecting a melt temperature signatures for at least two target regions of nucleic acid in a multiplex amplification reaction according to a method of any one of claims 1-24, the cartridge comprising: a) a cartridge body comprising a plurality' of chambers therein, wherein the plurality of chambers includes: i) a sample chamber having at least a fluid outlet in fluid communication with another chamber of the plurality; and ii) an optional lysis chamber in fluidic communication with the sample chamber, optionally wherein the sample chamber and lysis chamber are the same; b) a reaction vessel fluidically coupled to the plurality of chambers of the cartridge body and configured for: i) amplification of nucleic acid and ii) detection and identification of a plurality of amplification products via realtime PCR; c) a filter disposed in a fluidic path between the lysis chamber, if present, or the sample chamber, and the reaction vessel, and d) sets of primers and sets of two or more probes for detecting each target region, wherein each of the two or more probes in a set comprises a detectable label that emits light at the same wavelength.

26. The cartridge of claim 25, comprising a lysis chamber, wherein the lysis chamber comprises one or more lysis reagents for releasing nucleic acid.

27. The cartridge of any one of claim 25-26, wherein the reaction vessel comprises one or more reaction chambers for amplification and detection of the amplification products.

28. The cartridge of any one of claim 25-27, wherein the reaction vessel comprises one reaction chamber for amplification and detection of the amplification products.

29. The cartridge of any one of claim 25-27, wherein each reaction chamber is configured to detect a single amplification product.

30. The cartridge of any one of claim 25-27, wherein each reaction chamber is configured to detect a plurality⁷ of amplification products.

31. The cartridge of any one of claim 25-30, wherein the cartridge is a Clinical Laboratory Improvement Amendments (CLIA)-compliant cartridge.

32. The cartridge of any one of claim 25-31, wherein the cartridge is configured to cany' out non-isothermal amplification, optionally by thermal cycling or temperature oscillation, preferably by real-time PCR amplification.

33. A kit for detecting melt temperature signatures for at least two target regions of nucleic acid in a multiplex amplification reaction according to a method of any one or claims 1 -24, the kit comprising: sets of primers and sets of two or more probes for detecting each target region, wherein each of the two or more probes in the set comprises a detectable label that emits light at the same wavelength.