Respiratory tract biomarker combinations

Abstract

The present disclosure provides a set of primers and optional probes for identifying the presence of alpha-coronavirus, beta-coronavirus, SARS-CoV-2, adenovirus, chlamydia pneumoniae, influenza A, influenza B, metapneumovirus, rhinovirus / enterovirus, mycoplasma, Bordetella, parainfluenza, and respiratory syncytial virus (RSV), the present invention relates to nucleic acid probes, which may be included with a cartridge (e.g., in a kit) or in a cartridge, for automated detection of these pathogens by nucleic acid amplification. The disclosure also provides related detection methods, as well as kits, systems, and kits useful in such methods.

Description

[0001] Cross Reference to Related Applications

[0002] This application claims the benefit of U.S. Provisional Application No. 63 / 442,070, filed January 30, 2023, and U.S. Provisional Application No. 63 / 534,764, filed August 25, 2023, each of which is hereby incorporated by reference in its entirety.

[0003] TECHNICAL FIELD

[0004] The present invention relates generally to the field of detecting bacterial and / or viral respiratory pathogens.

[0005] BACKGROUND

[0006] Respiratory tract infections (RTIs) are one of the most common and important problems in clinical medicine worldwide. Approximately 90% of upper respiratory tract infections and approximately 30% of lower respiratory tract infections are caused by viruses. These lead to a variety of patient illnesses, including acute bronchitis, the common cold, influenza, and respiratory distress syndrome. Most of these patient illnesses are difficult to define because the manifestations associated with RTIs often overlap and their etiology is similar as well.

[0007] Many different types of pathogens cause RTIs, leading to both upper and lower RTIs, which can be localized respiratory tract infections or systemic infections. The most severe cases of RTIs occur in children under 5 years of age and in older individuals with low immune function. The main symptoms of RTIs include cough, congestion, runny nose, wheezing, headache, fever, and myalgia. The symptoms of bacterial infections and viral infections are similar, which makes it difficult to make a diagnosis without performing confirmatory diagnostic testing. Without the correct diagnosis and treatment of respiratory tract infections, many RTIs lead to pneumonia. Pneumonia accounts for 16% of all deaths in children under 5 years of age, taking 920136 children’s lives in 2015 alone. Pneumonia is the single largest infectious cause of child mortality worldwide. Pneumonia is a form of acute respiratory infection in which the alveolar sacs are filled with pus and fluid, causing painful breathing and difficulty exchanging oxygen. The presenting symptoms of viral pneumonia and bacterial pneumonia are similar, but require very different treatment regimens. Identifying the pathogen is important for doctors to provide proper patient management and treatment.

[0008] Respiratory viruses and bacteria have traditionally been detected by cell culture. Virus culture is slow (incubation times of at least one week) and requires a high level of operator experience. Antigen detection methods, such as enzyme-linked immunosorbent assays (ELISA), immunochromatographic (lateral flow) assays, and immunofluorescence assays (IFA) are commonly used to detect most respiratory viruses, but these methods suffer from lack of sensitivity. For bacterial respiratory infections, Bordetella species grow on appropriate agar, but more slowly than most other bacteria. For example, it is recommended that cultures be incubated for 10 days to improve recovery. Cultured isolates of Bordetella require rapid transport and specialized media to maintain the viability of the bacteria. Serological methods are occasionally used to diagnose pertussis, but there are no FDA-approved tests, and results can be difficult to interpret, leading to poor sensitivity and specificity. Mycoplasma pneumoniae can be recovered in specialized culture systems, but requires weeks of incubation, making it nearly useless for diagnosing infection. Molecular detection of IgM and serological tests are commonly used methods for diagnosing M. pneumoniae infection, and many IgM serological methods suffer from poor specificity. Chlamydia pneumoniae can be recovered in cell culture, but tests are slow, require expertise, and are not available in most laboratories. A few reference laboratories offer a serological method (IFA), but results are slow and often only provide retrospective information.

[0009] Molecular methods can provide higher sensitivity and faster time to result than culture. However, current nucleic acid amplification methods have limitations because the amplification reaction and signal detection require a controlled environment and precise measurements with expensive instrumentation. As a result, the methods are often cost prohibitive for use in point-of-care settings. Additionally, some methods are not optimized for detecting multiple target nucleic acids in a single patient sample. Detection of multiple targets can be achieved by signal multiplexing (fluorescence spectral multiplexing, electrochemical detector arrays) in a single tube reaction, physically separating multiple reactions into unique reaction vessels, or a combination thereof. Physically separating multiple reactions into unique reaction vessels can lead to erroneous results due to differences in sampling or assay conditions during the reaction, which can interfere with the work of differential diagnosis. Multiplexing can overcome some of these difficulties, but presents technical challenges of its own, particularly when trying to assay more than a few pathogens in a single reaction mixture. However, since the early 2000s, DNA detection technology has split into large-scale multiplexed but slow systems (next-generation sequencing (NGS) and microarrays), or rapid assays with limited multiplexing capability (quantitative PCR (qPCR) and isothermal amplification).

[0010] Further, in the case of CLIA-exempt tests, users can simply follow no more than three simple steps to simultaneously interrogate a panel of target nucleic acids using a single patient sample. Rapid physical separation of the sample into discrete chambers becomes infeasible for CLIA-exempt tests unless complex equipment or a one-time automated process is used. Fluorescence spectral multiplexing can reduce the number of unique reactions required to detect a panel of target nucleic acids, but spectrally multiplexed LAMP reactions require a significant sacrifice in assay speed or signal strength, which weakens their prospects for successful application in point-of-care testing.

[0011] In some embodiments, the methods, compositions, and apparatuses presented herein use a closed and affordable instrument, enabling rapid, sensitive, qualitative, and optionally quantitative detection of many target nucleic acids (DNA and RNA) in a single sample.

[0012] SUMMARY

[0013] The various embodiments contemplated herein can include, but are not necessarily limited to, one or more of the following:

[0014] Embodiment 1 : A cartridge for detecting and / or identifying viral and bacterial respiratory pathogens in a sample, the cartridge comprising: a cartridge body comprising a plurality of chambers therein, wherein the plurality of chambers comprises: a sample chamber having at least a fluidic outlet in fluid communication with another of the plurality of chambers; and optionally a lysis chamber in fluid communication with the sample chamber, optionally wherein the sample chamber and lysis chamber are the same chamber; a reaction vessel fluidically connected to the plurality of chambers of the cartridge body and configured for: i) amplification of nucleic acids and ii) detection and identification of a plurality of amplification products via real-time PCR, melt curve analysis, or a combination thereof; a filter disposed in the fluidic path between the lysis chamber, if present, or the sample chamber and the reaction vessel, and a set of primers and optionally probes for detecting and / or identifying the presence of alpha-coronavirus, beta-coronavirus, SARS-CoV-2, adenovirus, Chlamydia pneumoniae, influenza A, influenza B, metapneumovirus, rhinovirus / enterovirus, mycoplasma, bordetella, parainfluenza, and respiratory syncytial virus (RSV).

[0015] Embodiment 2: The cartridge of embodiment 1, wherein the lysis chamber comprises one or more lysis reagents for releasing nucleic acids.

[0016] Embodiment 3: The cartridge of embodiment 1 or embodiment 2, wherein the sample chamber and lysis chamber are the same chamber.

[0017] Embodiment 4: The cartridge of any one of embodiments 1-3, wherein the reaction vessel comprises one or more reaction chambers for detecting a plurality of amplification products.

[0018] Embodiment 5: The cartridge of any one of embodiments 1-4, wherein the reaction vessel comprises one reaction chamber.

[0019] Embodiment 6: The cartridge of embodiment 4, wherein each reaction chamber is configured to detect a single amplification product.

[0020] Embodiment 7: The cartridge of embodiment 4, wherein each reaction chamber is configured to detect a plurality of amplification products.

[0021] Embodiment 8: The cartridge of embodiment 7, wherein each reaction chamber is configured to detect amplification products from a- coronavirus, b-coronavirus, SARS-CoV-2, adenovirus, Chlamydia pneumoniae, influenza A, influenza B, metapneumovirus, rhinovirus / enterovirus, mycoplasma, bordetella, parainfluenza, and respiratory syncytial virus (RSV).

[0022] Embodiment 9: The cartridge of embodiment 7 or embodiment 8, wherein each reaction chamber is configured to simultaneously detect and identify at least 10 amplification products via real-time PCR.

[0023] Embodiment 10: The cartridge of embodiment 7 or embodiment 8, wherein each reaction chamber is configured to simultaneously detect and identify up to 10 amplification products via melt curve analysis.

[0024] Embodiment 11: The cartridge of any one of embodiments 1-10, wherein the cartridge is a Clinical Laboratory Improvement Amendments (CLIA)-compliant cartridge.

[0025] Embodiment 12: The cartridge of any one of embodiments 1-11, wherein the cartridge is configured to perform isothermal amplification.

[0026] Embodiment 13: The cartridge of any one of embodiments 1-11, wherein the cartridge is configured to perform non-isothermal amplification, optionally by thermal cycling or temperature oscillation.

[0027] Embodiment 14: A method for detecting and / or identifying viral and bacterial respiratory pathogens in a sample, the method comprising: a) contacting nucleic acids from a sample with a set of primers and optional probes for detecting the presence of alpha-coronavirus, beta-coronavirus, SARS-CoV-2, adenovirus, Chlamydia pneumoniae, influenza A, influenza B, metapneumovirus, rhinovirus / enterovirus, mycoplasma, Bordetella, parainfluenza, and respiratory syncytial virus (RSV); b) subjecting the nucleic acids, primers, and optional probes to amplification conditions followed by a melting curve assay; c) detecting the presence of any amplification product(s) via real-time PCR and melting curve analysis; and d) differentially identifying the presence of viral and / or bacterial respiratory pathogens in the sample based on the detection of amplification product(s) or the absence thereof, respectively, or determining the absence of viral or bacterial pathogens detectable using the primer set.

[0028] Embodiment 15: The method of Embodiment 14, wherein the method further comprises administering a treatment regimen to the subject based on the detection of the presence of at least one of the viral or bacterial respiratory pathogens and / or the severity of the infection.

[0029] Embodiment 16: The method of Embodiment 15, wherein the treatment regimen is selected from one or more of antibiotic administration, antiviral drug administration, mechanical ventilation, invasive monitoring, sedation, admission to intensive care, surgical intervention, last drug resort, or hospitalization.

[0030] Embodiment 17: The method of any one of Embodiments 14-16, wherein one or more of the bacterial respiratory pathogens is identified via melting curve analysis.

[0031] Embodiment 18: The method according to any one of Embodiments 14-16, wherein the viral and / or bacterial respiratory pathogens identified via melting curve analysis comprise metapneumovirus, influenza A, Mycoplasma pneumoniae, Chlamydia pneumoniae, Bordetella parapertussis, and Bordetella pertussis.

[0032] Embodiment 19: The method of any one of Embodiments 14-18, wherein: a) the contacting of nucleic acids from a sample with a set of primers and optional probes comprises: placing the sample in a cartridge comprising a cartridge body having a plurality of chambers in fluid communication, a reaction vessel having one or more reaction chambers and configured for amplification and detection of nucleic acids, a fluidic path between the plurality of chambers and the reaction vessel, and a filter in the fluidic path; and lysing cells in the sample with one or more lysis reagents present within at least one of the plurality of chambers if the sample comprises cells; b) the subjecting of the nucleic acids, primer pairs, and optional probes to amplification conditions comprises amplifying the nucleic acids with the primers and probes in solution present within the reaction vessel.

[0033] Embodiment 20: The method of embodiment 19, wherein the amplification is isothermal amplification.

[0034] Embodiment 21 : The method of embodiment 19, wherein the amplification is non-isothermal amplification, preferably non-isothermal amplification by thermal cycling or temperature oscillation.

[0035] Embodiment 22: The method of any one of embodiments 19-21, wherein the detecting is performed in more than one reaction chamber.

[0036] Embodiment 23: The method of any one of embodiments 19-21, wherein the detecting is performed in a single reaction chamber.

[0037] Embodiment 24: The method of embodiment 22 or embodiment 23, wherein each reaction chamber simultaneously detects at least 10 amplification products via real-time PCR.

[0038] Embodiment 25: The method of any one of embodiments 22-24, wherein each reaction chamber simultaneously detects up to 10 amplification products via melting curve analysis.

[0039] Embodiment 26: The method of any one of embodiments 14-25, wherein the sample is a respiratory sample selected from the group consisting of nasopharyngeal swab (NP), oropharyngeal swab (OP), nasal swab (NS), respiratory mucus sample, respiratory tissue sample, respiratory cell sample, saliva sample, sputum sample, or a combination thereof.

[0040] Embodiment 27: The method of any one of embodiments 14-25, wherein the sample is a wastewater sample.

[0041] Embodiment 28: The method of any one of embodiments 14-27, wherein the detecting and / or identifying is done at the same facility where the sample is collected from the subject.

[0042] Embodiment 29: The method of any one of embodiments 14-28, wherein the method is a point-of-care method.

[0043] Embodiment 30: The method of any one of embodiments 14-29, wherein the method is performed at a hospital, emergency center, emergency room, doctor’s office, health clinic, or at home.

[0044] Embodiment 31 : The method of any one of embodiments 14-30, wherein the method is a Clinical Laboratory Improvement Amendments (CLIA)-waived test.

[0045] Embodiment 32: The method of any one of embodiments 19-30, wherein the cartridge is a Clinical Laboratory Improvement Amendments (CLIA)-compliant cartridge, is operated pursuant to CLIA, is operated by a CLIA-compliant laboratory, or is operated at a CLIA-compliant site.

[0046] Embodiment 33: The method of any one of embodiments 19-32, wherein the method is performed to distinguish between a virulent pathogen and a weak virulent pathogen.

[0047] Embodiment 34: A kit comprising a cartridge comprising: a set of primers and optionally a probe for detecting the presence of an alpha-coronavirus, a beta-coronavirus, SARS-CoV-2, an adenovirus, Chlamydia pneumoniae, influenza A, influenza B, metapneumovirus, rhinovirus / enterovirus, mycoplasma, bordetella, parainfluenza, RSV, or a combination thereof; and optionally comprising one or more lysis reagents.

[0048] Embodiment 35: The cartridge of any one of embodiments 1-13, the method of any one of embodiments 14-33, or the kit of embodiment 34, wherein: the primers and optional (multiple) probes for detecting an alpha-coronavirus are capable of hybridizing to a spike glycoprotein gene of an alpha-coronavirus; the primers and optional (multiple) probes for detecting a beta-coronavirus are capable of hybridizing to an ORF1a gene of a beta-coronavirus type A; the primers and optional (multiple) probes for detecting a SARS-CoV-2 coronavirus are capable of hybridizing to an E gene, an N gene, an RDRP gene, or a combination thereof, of a SARS-CoV-2 coronavirus; the primers and optional (multiple) probes for detecting an adenovirus are capable of hybridizing to a hexon protein gene of an adenovirus type A, type B, type C, type D, and type E; the primers and optional (multiple) probes for detecting a chlamydia are capable of hybridizing to an ompA gene of Chlamydia pneumoniae; the primers and optional (multiple) probes for detecting an influenza A are capable of hybridizing to an HA gene, an MP gene, a PA gene, a PB gene, or a combination thereof, of an influenza A; the primers and optional (multiple) probes for detecting an influenza B are capable of hybridizing to an MP gene, an NS gene, or a combination thereof, of an influenza B; the primers and optional (multiple) probes for detecting a metapneumovirus are capable of hybridizing to an N gene of a human metapneumovirus; the primers and optional (multiple) probes for detecting a rhinovirus / enterovirus are capable of hybridizing to a 5'UTR gene of a rhinovirus / enterovirus; the primers and optional (multiple) probes for detecting a mycoplasma are capable of hybridizing to a P1 adhesion protein gene of a mycoplasma; the primers and optional (multiple) probes for detecting a Bordetella are including primers and optional (multiple) probes capable of hybridizing to an IS1001 gene of Bordetella parapertussis; the primers and optional (multiple) probes for detecting a Bordetella further include primers and optional (multiple) probes capable of hybridizing to an IS481 gene of Bordetella pertussis; the primers and optional (multiple) probes for detecting a parainfluenza are capable of hybridizing to a polymerase (L) protein gene, a nucleocapsid (NP) protein gene, or a combination thereof, of a parainfluenza type 1, type 2, type 3, and type 4; and the primers and optional (multiple) probes for detecting an RSV are capable of hybridizing to an RSV A gene, an RSV B gene, or a combination thereof.

[0049] Embodiment 36: The cartridge of any one of embodiments 1-13, the method of any one of embodiments 14-33, the kit of embodiment 34, or the cartridge, method, or kit of embodiment 35, wherein the primers and optional probes are configured to detect their target nucleic acid sequences in a single reaction mixture comprising 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, or 35 different primer pairs and optional probes.

[0050] Embodiment 37: The cartridge, method, or kit of embodiment 35 or embodiment 36, wherein the primers and / or probes capable of hybridizing to the ORF la gene of a beta-coronavirus comprise: at least one specific primer pair and optional probe specific for the ORF la gene of CoV-OC43; and / or at least one specific primer pair and optional probe specific for the ORF la gene of CoV-HKUl.

[0051] Embodiment 38: The cartridge, method, or kit of any one of embodiments 35-37, wherein the set of primers and optional probes capable of hybridizing to the spike glycoprotein gene of an alpha-coronavirus comprise: at least one specific primer pair and optional probe specific for the S gene of CoV-229E; and / or at least one specific primer pair and optional probe specific for the S gene of CoV-NL63.

[0052] Embodiment 39: The cartridge, method, or kit of any one of embodiments 35-38, wherein, when present: the primer and optional probe(s) capable of selective hybridization to the ORF la gene of CoV-OC43 comprise a sequence identical or complementary to at least 15 contiguous nucleotides of SEQ ID NO: 24; the primer and optional probe(s) capable of selective hybridization to the ORF la gene of CoV-HKUl comprise a sequence identical or complementary to at least 15 contiguous nucleotides of SEQ ID NO: 28; the primer and optional probe(s) capable of selective hybridization to the S gene of CoV-229E comprise a sequence identical or complementary to at least 15 contiguous nucleotides of SEQ ID NO: 20; the primer and optional probe(s) capable of selective hybridization to the S gene of CoV-NL63 comprise a sequence identical or complementary to at least 15 contiguous nucleotides of SEQ ID NO: 32; the primer and optional probe(s) capable of selective hybridization to the N gene of SARS-CoV-2 comprise a sequence identical or complementary to at least 15 contiguous nucleotides of SEQ ID NO: 96; the primer and optional probe(s) capable of selective hybridization to the RDRP gene of SARS-CoV-2 comprise a sequence identical or complementary to at least 15 contiguous nucleotides of SEQ ID NO: 104; the primer and optional probe(s) capable of selective hybridization to the E gene of SARS-CoV-2 comprise a sequence identical or complementary to at least 15 contiguous nucleotides of SEQ ID NO: 100; the primer and optional probe(s) capable of selective hybridization to the hexon protein gene of Adenovirus types A, B, C, D, and E comprise a sequence identical or complementary to at least 15 contiguous nucleotides of SEQ ID NO: 4; the primer and optional probe(s) capable of selective hybridization to the ompA gene of Chlamydia pneumoniae comprise a sequence identical or complementary to at least 15 contiguous nucleotides of SEQ ID NO: 16; the primer and optional probe(s) capable of selective hybridization to the PB gene of Influenza A comprise a sequence identical or complementary to at least 15 contiguous nucleotides of SEQ ID NO: 40; the primer and optional probe(s) capable of selective hybridization to the MP gene of Influenza A comprise a sequence identical or complementary to at least 15 contiguous nucleotides of SEQ ID NO: 36; the primer and optional probe(s) capable of selective hybridization to the PA gene of Influenza A comprise a sequence identical or complementary to at least 15 contiguous nucleotides of SEQ ID NO: 44; the primer and optional probe(s) capable of selective hybridization to the H1 gene of Influenza A comprise a sequence identical or complementary to at least 15 contiguous nucleotides of SEQ ID NO: 48;Primers and optional probe(s) capable of selectively hybridizing to the MP gene of influenza B comprise a sequence identical or complementary to at least 15 contiguous nucleotides of SEQ ID NO: 52, primers and optional probe(s) capable of selectively hybridizing to the NS gene of influenza B comprise a sequence identical or complementary to at least 15 contiguous nucleotides of one or more of SEQ ID NO: 56; primers and optional probe(s) capable of selectively hybridizing to the N gene of human metapneumovirus comprise a sequence identical or complementary to at least 15 contiguous nucleotides of SEQ ID NO: 60; primers and optional probe(s) capable of selectively hybridizing to the 5'UTR gene of rhinovirus / enterovirus comprise a sequence identical or complementary to at least 15 contiguous nucleotides of SEQ ID NO: 84; primers and optional probe(s) capable of selectively hybridizing to the P1 adhesion protein gene of Mycoplasma pneumoniae comprise a sequence identical or complementary to at least 15 contiguous nucleotides of SEQ ID NO: 64; primers and optional probe(s) capable of selectively hybridizing to the IS1001 gene of Bordetella parapertussis comprise a sequence identical or complementary to at least 15 contiguous nucleotides of SEQ ID NO: 8; primers and optional probe(s) capable of selectively hybridizing to the IS481 gene of Bordetella pertussis comprise a sequence identical or complementary to at least 15 contiguous nucleotides of SEQ ID NO: 12; primers and optional probe(s) capable of selectively hybridizing to the polymerase (L) protein gene of parainfluenza types 1 and 3 comprise a sequence identical or complementary to at least 15 contiguous nucleotides of one or more of SEQ ID NO: 68 and 76, respectively; primers and optional probe(s) capable of selectively hybridizing to the nucleocapsid (NP) protein gene of parainfluenza types 2 and 4 comprise a sequence identical or complementary to at least 15 contiguous nucleotides of one or more of SEQ ID NO: 72 and 80, respectively; primers and optional probe(s) capable of selectively hybridizing to the RSV A gene comprise a sequence identical or complementary to at least 15 contiguous nucleotides of SEQ ID NO: 88; and primers and optional probe(s) capable of selectively hybridizing to the RSV B gene comprise a sequence identical or complementary to at least 15 contiguous nucleotides of SEQ ID NO: 92.

[0053] Embodiment 40: The cartridge, method, or kit of any one of embodiments 35-38, wherein, when present: the primer and optional probe(s) capable of selective hybridization to the ORF la gene of CoV-OC43 comprise a sequence identical or complementary to at least 15 contiguous nucleotides of one or more of SEQ ID NOs: 21, 22, and 23; the primer and optional probe(s) capable of selective hybridization to the ORF la gene of CoV-HKUl comprise a sequence identical or complementary to at least 15 contiguous nucleotides of one or more of SEQ ID NOs: 25, 26, and 27; the primer and optional probe(s) capable of selective hybridization to the S gene of CoV-229E comprise a sequence identical or complementary to at least 15 contiguous nucleotides of one or more of SEQ ID NOs: 17, 18, and 19; the primer and optional probe(s) capable of selective hybridization to the S gene of CoV-NL63 comprise a sequence identical or complementary to at least 15 contiguous nucleotides of one or more of SEQ ID NOs: 29, 30, and 31; the primer and optional probe(s) capable of selective hybridization to the N gene of SARS-CoV-2 comprise a sequence identical or complementary to at least 15 contiguous nucleotides of one or more of SEQ ID NOs: 93, 94, and 95; the primer and optional probe(s) capable of selective hybridization to the RDRP gene of SARS-CoV-2 comprise a sequence identical or complementary to at least 15 contiguous nucleotides of one or more of SEQ ID NOs: 101, 102, and 103; the primer and optional probe(s) capable of selective hybridization to the E gene of SARS-CoV-2 comprise a sequence identical or complementary to at least 15 contiguous nucleotides of one or more of SEQ ID NOs: 97, 98, and 99; the primer and optional probe(s) capable of selective hybridization to the hexon protein gene of Adenovirus types A, B, C, D, and E comprise a sequence identical or complementary to at least 15 contiguous nucleotides of one or more of SEQ ID NOs: 1, 2, and 3; the primer and optional probe(s) capable of selective hybridization to the ompA gene of Chlamydia pneumoniae comprise a sequence identical or complementary to at least 15 contiguous nucleotides of one or more of SEQ ID NOs: 13, 14, and 15; the primer and optional probe(s) capable of selective hybridization to the PB gene of Influenza A comprise a sequence identical or complementary to at least 15 contiguous nucleotides of one or more of SEQ ID NOs: 37, 38, and 39; the primer and optional probe(s) capable of selective hybridization to the MP gene of Influenza A comprise a sequence identical or complementary to at least 15 contiguous nucleotides of one or more of SEQ ID NOs: 33, 34, and 35;The primers and optional probe(s) capable of selectively hybridizing to the PA gene of influenza A comprise a sequence identical or complementary to at least 15 contiguous nucleotides of one or more of SEQ ID NOs:41, 42, and 43; the primers and optional probe(s) capable of selectively hybridizing to the H1 gene of influenza A comprise a sequence identical or complementary to at least 15 contiguous nucleotides of SEQ ID NOs:45, 46, and 47; the primers and optional probe(s) capable of selectively hybridizing to the MP gene of influenza B comprise a sequence identical or complementary to at least 15 contiguous nucleotides of one or more of SEQ ID NOs:49, 50, and 51, the primers and optional probe(s) capable of selectively hybridizing to the NS gene of influenza B comprise a sequence identical or complementary to at least 15 contiguous nucleotides of one or more of SEQ ID NOs:53, 54, and 55; the primers and optional probe(s) capable of selectively hybridizing to the N gene of human metapneumovirus comprise a sequence identical or complementary to at least 15 contiguous nucleotides of one or more of SEQ ID NOs:57, 58, and 59; the primers and optional probe(s) capable of selectively hybridizing to the 5'UTR gene of rhinovirus / enterovirus comprise a sequence identical or complementary to at least 15 contiguous nucleotides of one or more of SEQ ID NOs:81, 82, and 83; the primers and optional probe(s) capable of selectively hybridizing to the P1 adhesin protein gene of Mycoplasma pneumoniae comprise a sequence identical or complementary to at least 15 contiguous nucleotides of one or more of SEQ ID NOs:61, 62, and 63; the primers and optional probe(s) capable of selectively hybridizing to the IS1001 gene of Bordetella parapertussis comprise a sequence identical or complementary to at least 15 contiguous nucleotides of one or more of SEQ ID NOs:5, 6, and 7; the primers and optional probe(s) capable of selectively hybridizing to the IS481 gene of Bordetella pertussis comprise a sequence identical or complementary to at least 15 contiguous nucleotides of one or more of SEQ ID NOs:9, 10, and 11; the primers and optional probe(s) capable of selectively hybridizing to the polymerase (L) protein gene of parainfluenza types 1 and 3 comprise a sequence identical or complementary to at least 15 contiguous nucleotides of one or more of SEQ ID NOs:68 and 76, respectively; the primers and optional probe(s) capable of selectively hybridizing to the nucleocapsid (NP) protein gene of parainfluenza types 2 and 4 comprise a sequence identical or complementary to at least 15 contiguous nucleotides of one or more of SEQ ID NOs:72 and 80, respectively; the primers and optional probe(s) capable of selectively hybridizing to the RSV A gene comprise a sequence identical or complementary to at least 15 contiguous nucleotides of one or more of SEQ ID NOs:85, 86, and 87;and the primer and optional (s) probe capable of selectively hybridizing to the RSV B gene comprises a sequence identical or complementary to at least 15 contiguous nucleotides of one or more of SEQ ID NOs: 89, 90, and 91.

[0054] Embodiment 41 : The cartridge of any one of embodiments 1-13, the method of any one of embodiments 14-33, the kit of embodiment 34, or the cartridge, method, or kit of any one of embodiments 35-40, wherein at least one of the primers and optional probes comprises a detectable label.

[0055] Embodiment 42: The cartridge, method, or kit of embodiment 41, wherein at least one probe comprises a fluorescent dye and a quencher molecule.

[0056] Embodiment 43: The cartridge of any one of embodiments 1-13, the method of any one of embodiments 14-33, the kit of embodiment 34, or the cartridge, method, or kit of any one of embodiments 35-42, wherein the cartridge or kit further comprises or the method further employs a primer pair specific for 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.

[0057] Embodiment 44: The cartridge of any one of embodiments 1-13, the method of any one of embodiments 19-33, or the kit of embodiment 34, wherein the cartridge or kit facilitates and / or the method comprises detecting viral and / or bacterial respiratory pathogens within a biological sample within 60 minutes, within 45 minutes, or within 30 minutes of collecting the sample from the subject.

[0058] Embodiment 45: The cartridge of any one of embodiments 2-13, the method of any one of embodiments 19-33, the kit of embodiment 34, or the cartridge, method, or kit of any one of embodiments 35-44, wherein the one or more lysis reagents provide simultaneous lysis of viral and bacterial cells.

[0059] Embodiment 46: The cartridge of any one of embodiments 2-13, the method of any one of embodiments 19-33, the kit of embodiment 34, or the cartridge, method, or kit of any one of embodiments 35-45, wherein the one or more lysis reagents comprise a chaotropic agent, a chelating agent, a buffer, and a detergent.

[0060] Embodiment 47: The cartridge, method, or kit of embodiment 46, wherein the chaotropic agent is selected from guanidinium thiocyanate, guanidinium hydrochloride, an alkali metal perchlorate, an alkali metal iodide, urea, formamide, or a combination thereof.

[0061] Embodiment 48: The cartridge of any one of embodiments 2-13, the method of any one of embodiments 19-33, the kit of embodiment 34, or the cartridge, method, or kit of any one of embodiments 35-47, wherein the one or more lysis reagents comprise a guanidine compound, sodium hydroxide, EDTA, a buffer, and a detergent.

[0062] Embodiment 49: The cartridge of any one of embodiments 1-13 or the method of any one of embodiments 19-33, wherein the filter is configured to bind nucleic acids to be analyzed.

[0063] Embodiment 50: The cartridge or method of embodiment 49, wherein the filter comprises glass fibers and optionally a polymeric binder, or the glass fibers are optionally modified with a DNA binding ligand, such as an alkyl amine, a cycloalkyl amine, an alkoxy amine, a polyamine moiety, an aryl amine, an intercalator, a DNA groove binder, a peptide, an amino acid, a protein, or a combination thereof.

[0064] Embodiment 51: The cartridge or method of embodiment 50, wherein the filter comprises a 500 micrometers to 2000 micrometers thick disc of glass fibers having a pore size of 0.2 micrometers to 1 micrometers.

[0065] Embodiment 52: The cartridge of any one of embodiments 1-13 or the method of any one of embodiments 19-33, wherein the filter is configured to bind unwanted substances and allow nucleic acids to pass through.

[0066] Embodiment 53: The cartridge of any one of embodiments 1-13, the method of any one of embodiments 19-33, or the kit of embodiment 34, wherein the cartridge further comprises a binding reagent, a wash reagent, an elution reagent, or a combination thereof.

[0067] Embodiment 54: The cartridge, method, or kit of embodiment 53, wherein the elution reagent comprises ammonia or an alkali hydroxide.

[0068] Embodiment 55: The cartridge, method, or kit of embodiment 53 or embodiment 54, wherein the elution reagent has a pH higher than about 9, higher than about 10, or higher than about 11.

[0069] Embodiment 56: The cartridge, method, or kit of any one of embodiments 53-55, wherein the elution reagent comprises a polyanion, which is optionally selected from carrageenan, a carrier nucleic acid, and i-carrageenan comprising KOH.

[0070] Embodiment 57: The cartridge of any one of embodiments 1-13 or the method of any one of embodiments 19-33, wherein the reaction vessel comprises up to 4 reaction chambers.

[0071] Embodiment 58: The cartridge of any one of embodiments 1-13 or the method of any one of embodiments 19-33, wherein at least one of the plurality of chambers comprises one or more lyophilized reagents.

[0072] Embodiment 59: The cartridge or method of embodiment 58, wherein the one or more lyophilized reagents are in the form of one or more microbeads.

[0073] Embodiment 60: The cartridge or method of embodiment 58 or embodiment 59, wherein the one or more lyophilized reagents are selected from primers, probes, salts, dNTPs, thermostable polymerases, reverse transcriptases, or combinations thereof.

[0074] Embodiment 61 : The cartridge or method of embodiment 60, wherein the one or more lyophilized reagents comprise lyophilized primers and probes.

[0075] Embodiment 62: The cartridge of any one of embodiments 1-13 or the method of any one of embodiments 19-33, wherein the reagents and components in the reaction vessel are in solution.

[0076] Embodiment 63: The cartridge or method of any one of embodiments 1-62, wherein the limit of detection (LOD) of the cartridge or method is greater than or equal to about 25 TCID50 / mL for adenovirus, greater than or equal to about 0.1 TCID50 / mL for coronavirus 229E, greater than or equal to about 100 copies / mL for coronavirus HKU1, greater than or equal to about 0.1 TCID50 / mL for coronavirus NL63, greater than or equal to about 0.1 TCID50 / mL for coronavirus OC43, greater than or equal to about 1500 IU / mL for SARS-CoV-2, greater than or equal to about 0.5 TCID50 / mL for human metapneumovirus (hMPV), greater than or equal to about 4 TCID50 / mL for human rhinovirus, greater than or equal to about 8 TCID50 / mL for enterovirus, greater than or equal to about 8 TCID50 / mL for parainfluenza type 1, greater than or equal to about 2 TCID50 / mL for parainfluenza type 2, greater than or equal to about 10 TCID50 / mL for parainfluenza type 3, greater than or equal to about 8 TCID50 / mL for parainfluenza type 4, greater than or equal to 16 TCID50 / mL for influenza A H3N2, greater than or equal to about 0.5 TCID50 / mL for influenza A H1N1, greater than or equal to about 4.4 TCID50 / mL for influenza B, greater than or equal to about 0.1 TCID50 / mL for respiratory syncytial virus (RSV) type A, greater than or equal to about 0.05 TCID50 / mL for respiratory syncytial virus (RSV) type B, greater than or equal to about 200 CFU / mL for Bordetella pertussis, greater than or equal to about 8 CFU / mL for Bordetella parapertussis, greater than or equal to about 15 CCU / mL for Mycoplasma pneumoniae, and / or greater than or equal to about 1 TCID50 / mL for Chlamydia pneumoniae.

[0077] Embodiment 64: A system for detecting a pathogen in a biological sample, the system comprising: a module having a receiving slot for receiving a cartridge of any one of embodiments 1-13, wherein the module includes one or more mechanisms within the receiving slot for manipulating a fluid sample within the cartridge, and an instrument interfaced with the reaction vessel; and a memory having recorded thereon programmable instructions specifically configured to operate the module according to a respiratory pathogen assay protocol to determine nucleic acid sequence signatures of alpha-coronavirus, beta-coronavirus, SARS-CoV-2, adenovirus, Chlamydia pneumoniae, influenza A, influenza B, metapneumovirus, rhinovirus / enterovirus, mycoplasma, Bordetella, parainfluenza, and respiratory syncytial virus (RSV).

[0078] Embodiment 65: The system of embodiment 64, wherein the module and / or system further comprises: a scanner or reader configured to read an identifier on the cartridge; wherein the instructions are configured to determine an applicable protocol based on the reading or scanning of the identifier; and wherein the system operates the module according to the applicable protocol based on input from the scanner or reader.

[0079] Embodiment 66: The system of any one of embodiments 64-65, wherein the module and / or system further comprises: a housing; a plurality of modules comprising the module, wherein the modules are substantially identical and configured to simultaneously perform an assay on a cartridge received therein.

[0080] Embodiment 67: The system of any one of embodiments 64-66, further comprising a networking platform for transmitting results derived from the operation of the module.

[0081] Embodiment 68: A method for identifying up to forty target nucleic acids in a biological sample collected from a subject, the method comprising: a) providing a sample cartridge comprising: i) a plurality of chambers, including: a lysis chamber having at least a fluidic outlet in fluid communication with another of the plurality of chambers, the lysis chamber adapted for mechanical lysis and chemical lysis to release nucleic acids from the biological sample; ii) lyophilized reagents and liquid reagents in two or more of the plurality of chambers, the lyophilized reagents comprising at least one primer set for amplification and detection of the target nucleic acid(s), and the liquid reagents including lysis buffer, binding reagents, and elution buffer; and iii) a reaction vessel fluidically connected to the plurality of chambers of the sample cartridge and configured for performing amplification of the target nucleic acids; b) contacting nucleic acids from the biological sample with at least one primer set, i) wherein each primer set comprises a forward primer, a reverse primer, and a probe specific for a target nucleic acid in the biological sample, and ii) wherein at least one of the forward primer or reverse primer within each set comprises one or more modified bases selected from an unstable base, a stable base, or a combination thereof; and iii) wherein the probe in each set comprises a detectable moiety for detection via real-time PCR or melt curve analysis; c) subjecting the nucleic acids, primers, and probes within the reaction vessel to amplification conditions, optionally followed by a melt curve assay, d) detecting the presence of any amplification product(s) via real-time PCR and optional melt curve analysis by an optical assembly, wherein the optical assembly comprises at least ten optical channels configured to simultaneously transmit excitation light beams within at least ten excitation wavelength ranges and to simultaneously detect emission light within at least ten emission wavelength ranges to and from the reaction vessel, respectively; and e) differentially identifying the presence of the target nucleic acid(s) in the sample based on detection of the amplification product(s) or absence thereof, respectively, wherein when more than 10 target nucleic acids are to be identified, one or more probes for detection via melt curve analysis and one or more probes for detection via real-time PCR are combined in a single optical channel.

[0082] Embodiment 69: The method of embodiment 68, wherein the lysis chamber, along with the lysis reagents, is adapted to lyse viruses, bacteria, fungi, protozoa, and epithelial cells present in the biological sample.

[0083] Embodiment 70: The method of embodiment 69, wherein the method does not include mechanical lysis of the biological sample.

[0084] Embodiment 71: The method of embodiment 69, wherein the method further includes mechanical lysis of the biological sample.

[0085] Embodiment 72: The method of any one of embodiments 68-71, wherein the biological sample is selected from a respiratory sample comprising a nasopharyngeal swab (NP) sample, an oropharyngeal swab (OP) sample, a nasal swab (NS) sample, a respiratory mucus sample, a respiratory tissue sample, or a respiratory cell sample; a sample comprising saliva, sputum, blood, plasma, serum, semen, spinal fluid, tissue, tears, urine, fecal matter; or a smear specimen, a vaginal swab sample, a vaginal mucus sample, a vaginal tissue sample, a vaginal cell sample, a bacterial culture, a mammalian cell culture, a viral culture, a human cell sample, a bacterial sample, an extracellular fluid sample, a pancreatic juice sample, a cell lysate, a PCR reaction mixture, an in vitro nucleic acid modification reaction mixture, or a combination thereof.

[0086] Embodiment 73: The method of any one of embodiments 68-72, wherein the target nucleic acid is selected from a viral pathogen, a bacterial pathogen, a protozoan pathogen, or a combination thereof.

[0087] Embodiment 74: The method of any one of embodiments 68-73, wherein the target nucleic acid is selected from a viral respiratory pathogen, a bacterial respiratory pathogen, or a combination thereof.

[0088] Embodiment 75: The method of any one of embodiments 68-74, wherein the target nucleic acid comprises a target nucleic acid from an alpha-coronavirus, a beta-coronavirus, SARS-CoV-2, an adenovirus, Chlamydia pneumoniae, influenza A, influenza B, metapneumovirus, rhinovirus / enterovirus, mycoplasma, bordetella, parainfluenza, respiratory syncytial virus (RSV), or a combination thereof.

[0089] Embodiment 76: The method of any one of embodiments 68-75, wherein the target nucleic acid comprises a target nucleic acid from an alpha-coronavirus, a beta-coronavirus, SARS-CoV-2, an adenovirus, Chlamydia pneumoniae, influenza A, influenza B, metapneumovirus, rhinovirus / enterovirus, mycoplasma, bordetella, parainfluenza, and respiratory syncytial virus (RSV).

[0090] Embodiment 77: The method of any one of embodiments 68-76, wherein the reaction vessel comprises up to 5 reaction chambers, each reaction chamber configured for amplification of up to forty target nucleic acids in solution.

[0091] Embodiment 78: The method of embodiment 77, wherein the reaction vessel comprises a single reaction chamber.

[0092] Embodiment 79: The method of any one of embodiments 77-78, wherein the optical assembly detects up to ten (10) emission wavelength ranges simultaneously in each amplification cycle via real-time PCR and up to ten (10) emission wavelength ranges simultaneously in each melt curve cycle in each reaction chamber.

[0093] Embodiment 80: The method of embodiment 79, wherein the method differentially identifies up to thirty (30) target nucleic acids in each melt curve assay by combining probes for multiple (up to three) target nucleic acids in each optical channel and defining a melting window for each target nucleic acid.

[0094] Embodiment 81: The method of any one of embodiments 68-80, wherein when two or more probes are combined in an optical channel, the probe(s) detected by the melt curve assay are configured to have a Tm lower than the annealing temperature.

[0095] Embodiment 82: The method of any one of embodiments 68-81, wherein when more than ten (10) target nucleic acids are to be identified, target nucleic acids from a virus are detected via real-time PCR and target nucleic acids from a bacteria are detected via a melt curve assay.

[0096] Embodiment 83: The method of any one of embodiments 68-82, wherein the destabilizing base is selected from a bulky steric base that reduces non-specific primer- primer interactions, a base that inhibits the melting temperature of double-stranded form primers, a base that inhibits unwanted primer-dimer extension, or a combination thereof.

[0097] Embodiment 84: The method of any one of embodiments 68-83, wherein the stabilizing base increases the melting temperature of double-stranded form primers or reduces secondary structure formation.

[0098] Embodiment 85: The method of any one of embodiments 68-84, comprising up to 40 primer sets.

[0099] Embodiment 86: The method of any one of embodiments 68-85, comprising at least 10 primer sets.

[0100] Embodiment 87: The method of any one of embodiments 68-85, comprising at least 14 primer sets.

[0101] Embodiment 88: The method of any one of embodiments 68-87, wherein the primer sets comprise the primers and probes of any one of embodiments 35-40.

[0102] Embodiment 89: The method of any one of embodiments 68-88, wherein the cartridge is further adapted to perform high-resolution melt detection, nested PCR, multiplex detection, or a combination thereof.

[0103] Embodiment 90: The method of any one of embodiments 68-89, wherein the method does not comprise nested PCR.

[0104] Embodiment 91 : A sample cartridge for identifying up to forty target nucleic acids in a biological sample collected from a subject, the cartridge comprising: a) a cartridge body comprising a plurality of chambers therein, wherein the plurality of chambers comprises: i) a sample chamber having at least a fluidic outlet in fluid communication with another of the plurality of chambers; ii) a lysis chamber in fluid communication with the sample chamber, wherein the lysis chamber is adapted for mechanical lysis and chemical lysis to release nucleic acids from the biological sample, optionally wherein the sample chamber and lysis chamber are the same chamber; i) a lyophilized reagent chamber comprising a lyophilized PCR master mix comprising at least one primer set for amplification and detection of a target nucleic acid, further: a) wherein each primer set comprises a forward primer, a reverse primer, and a probe specific for a target nucleic acid in the biological sample, and b) wherein at least one of the forward primer or reverse primer within each set comprises one or more modified bases selected from an unstable base, a stable base, or a combination thereof; and c) wherein the probe in each set comprises a detectable moiety for detection via real-time PCR or melt curve analysis; iii) one or more liquid reagent chambers comprising lysis buffer, binding reagents, and elution buffer; b) a reaction vessel fluidically connected to the plurality of chambers of the cartridge body and configured for amplification and melt curve assay of nucleic acids and adapted for detection of one or more amplification products via real-time PCR and melt curve analysis by an optical assembly, wherein the optical assembly comprises at least ten optical channels configured to simultaneously transmit excitation light beams in at least ten excitation wavelength ranges to the reaction vessel and simultaneously detect emission light in at least ten emission wavelength ranges from the reaction vessel; and c) a filter disposed in a fluidic path between the lysis chamber and the reaction vessel; and wherein when more than 10 target nucleic acids are to be identified, one or more probes for detection via melt curve analysis and one or more probes for detection via real-time PCR are combined in a single optical channel.

[0105] Embodiment 92: A diagnostic assay system for identifying up to forty target nucleic acids in a biological sample collected from a subject, the system comprising: a) a sample cartridge of Embodiment 93; b) a thermal assembly configured to bring nucleic acids, primers and probes within a reaction vessel of the sample chamber under amplification conditions, optionally followed by a melt curve assay; c) an optical assembly comprising at least ten optical channels and configured to simultaneously transmit excitation light beams within at least ten excitation wavelength ranges and to simultaneously detect emission light to and from the reaction vessel within at least ten emission wavelength ranges, respectively; d) an electronic device for i) receiving and processing signals detected by the optical assembly via real-time PCR and melt curve assay resulting from the presence of any amplification product(s) and ii) differentially identifying the presence of a target nucleic acid in the sample based on the detection of the amplification product(s), melt curve or absence thereof, or determining that no target nucleic acid is detectable, and iii) outputting real-time PCR results of the sample in the reaction vessel during or after amplification and melt results of the sample in the reaction vessel during or after melt probe assay, e) a communication interface in communication with the electronic device, the electronic device comprising a display screen with a user interface displaying one or more graphical elements allowing a user to input a selection corresponding to at least one target assay of a plurality of target assays aimed at determining the presence, absence or amount of at least one target nucleic acid in a biological sample. BRIEF DESCRIPTION OF DRAWINGS

[0107] FIG. 1 A-1C shows an overview of a sample cartridge with a valve assembly configured for performing different sample processing, including chemical lysis of targets, the cartridge being configured for PCR and optional integrated nucleic acid analysis of a respiratory biomarker panel, according to some embodiments of the application. FIG. 1 A shows a sample cartridge body with a reaction vessel, FIG. 1 B shows an exploded view of the sample cartridge, and FIG. 1 C shows components of the valve assembly.

[0108] FIG. 2 The various valve assemblies A, B, C, D are illustrated, each adapted for one or more types of target lysis, any of which can be used in a respective sample cartridge.

[0109] FIGS. 3A-3B Illustrative, but not limiting, embodiments of modules and systems (e.g., processing units) for PCR detection and / or quantification of a biomarker panel and optional integrated nucleic acid analysis are shown. FIG. 3AIllustrated is a module configured to receive and interact with a valve assembly of a cartridge to operate the cartridge to facilitate sample preparation and analysis. FIG. 3B Illustrated is a processing unit (e.g., an analytical testing unit) of a module that interacts with a fluid sample in a reaction vessel to facilitate sample processing and analytical testing (e.g., PCR and optional nucleic acid analysis) of a biomarker panel.

[0110] FIG. 4 Illustrated is an exploded view of a sample cartridge illustrating its main components, including a lid, a multi-chamber body, reaction vessels, a valve assembly, and a base, according to some embodiments. Chambers can be used for various processing of a sample (e.g., extraction, purification, deamidation, desulfonation).

[0111] FIGS. 5A-5B Shown are specialized components of a cartridge configured for a target assay panel, according to some embodiments. As shown, the specialized components are a valve assembly with a syringe for sample injection and multiple ports on the valve body that facilitate transport of a fluid sample between various processing chambers when the valve assembly is rotated.

[0112] FIG. 6 Shown are SARS-COV-2 PQC data for pre-change development lots. See Example 3.

[0113] FIG. 7 Shown are FLUA PQC data for pre-change development lots. See Example 3.

[0114] FIG. 8 Shown are RSV PQC data for pre-change development lots. See Example 3.

[0115] FIG. 9 Shown are RHINO PQC data for pre-change development lots. See Example 3.

[0116] FIG. 10 Shown are MYCO PQC data for pre-change development lots. See Example 3.

[0117] FIG. 11 Shown are Bordetella pertussis PQC data for pre-change development lots. See Example 3.

[0118] DETAILED DESCRIPTION

[0119] The present disclosure describes methods, compositions, devices, and systems that facilitate rapid detection of viral and bacterial respiratory pathogens in a selective and specific manner that is amenable to automation and can be used in point-of-care devices, enabling detection of the pathogen causing a respiratory infection in a subject so that appropriate treatment can be administered in a timely manner. The methods rely on nucleic acid amplification to detect selected respiratory pathogens, which entails contacting sample nucleic acids with primers and optionally probes that target viral or bacterial nucleic acids of each pathogen, subjecting the nucleic acids, primers, and optionally probes to amplification conditions, and detecting the presence of any amplification product(s) to differentially identify the presence of viral and / or bacterial respiratory pathogens in the sample, or to determine the absence of a viral or bacterial pathogen in the sample that can be detected using the primer set.

[0120] DEFINITIONS

[0121] The definitions of the terms used in the claims and the specification are as follows, unless otherwise stated.

[0122] The term "nucleic acid" refers to a nucleotide polymer, unless otherwise limited, including analogs of naturally occurring nucleotides that can function in a similar manner (e.g., hybridize) as naturally occurring nucleotides.

[0123] The term nucleic acid encompasses DNA or RNA in any form, including, for example, genomic DNA; complementary DNA (cDNA), which is a DNA representation of mRNA, typically obtained by reverse transcription of messenger RNA (mRNA) or viral RNA or by amplification; a DNA molecule synthesized or produced by amplification; mRNA; and non-coding RNA.

[0124] The term nucleic acid encompasses double- or triple-stranded nucleic acid complexes, as well as single-stranded molecules. In double- or triple-stranded nucleic acid complexes, the nucleic acid strands need not be coextensive (i.e., double-stranded nucleic acids need not be double-stranded along their entire length).

[0125] The term nucleic acid also encompasses any modifications thereof, such as by methylation and / or by capping. Nucleic acid modifications can include the addition of chemical groups that confer additional charge, polarity, hydrogen bonding, electrostatic interactions, and functionality to individual nucleic acid bases or to the entire nucleic acid. Such modifications can include base modifications, such as 2'-position sugar modifications, 5-position pyrimidine modifications, 8-position purine modifications, cytosine exocyclic amine modifications, 5-bromo-uracil substitutions, sugar-phosphate backbone modifications, unconventional base pairing combinations such as isobasises isocytidine and isoguanine, and the like.

[0126] More specifically, in some embodiments, nucleic acids can include polydeoxyribonucleotides (containing 2-deoxy-D-ribose), polydeoxyribonucleotides (containing D-ribose), and any other type of N- or C-glycoside of a purine or pyrimidine base, as well as other polymers containing non-nucleotidic backbones, such as polyamides (e.g., peptide nucleic acids (PNAs)) and poly-morpholino polymers (see, e.g., Summerton and Weller (1997) “Morpholino Antisense Oligomers: Design, Preparation, and Properties,” Antisense & Nucleic Acid Drug Dev. 7: 181-195; Okamoto et al. (2002) “Development of electrochemically gene-analyzing method using DNA-modified electrodes,” Nucleic Acids Res. Supplement No. 2: 171-172), as well as other synthetic sequence-specific nucleic acid polymers, provided that the polymers contain nucleobases in a configuration that enables base pairing and base stacking, such as found in DNA and RNA. The term nucleic acid also encompasses locked nucleic acids (LNAs), which are described in U.S. Pat. Nos. 6,794,499, 6,670,461, 6,262,490, and 6,770,748, the disclosures of which regarding LNAs are incorporated herein by reference in their entireties.

[0127] The nucleic acid(s) can be derived from a completely chemical synthesis method, such as solid-phase mediated chemical synthesis, from a biological source, such as by isolation from any species that produces nucleic acids, or from a method involving manipulation of nucleic acids by molecular biology tools, such as DNA replication, PCR amplification, reverse transcription, or a combination of these methods.

[0128] As used herein, the term “gene” encompasses coding sequences, introns, and any associated control sequences involved in the expression of a coding sequence.

[0129] In the context of two or more amino acid sequences or nucleotide sequences, the term “sequence identity” refers to two or more sequences that are the same or have a specified percentage of identical amino acid residues or nucleotides, as measured using a sequence comparison algorithm or by visual inspection when compared and aligned for maximum match.

[0130] For purposes of sequence comparison, typically one sequence acts as the reference sequence, to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are input into a computer, subsequence coordinates are designated if necessary, and sequence algorithm parameters are designated. The sequence comparison algorithm then calculates the percent sequence identity for the test sequence(s) relative to the reference sequence, based on the designated sequence algorithm parameters. Sequence alignments for comparison can be performed using BLAST set to default parameters.

[0131] As used herein, the term "complementary" refers to the ability of nucleotides to pair precisely between two nucleotides; that is, if the nucleotide at a given position of a nucleic acid can hydrogen bond with the nucleotide of another nucleic acid to form a canonical base pair, then the two nucleic acids are considered to be complementary to each other at that position. Complementarity between two single-stranded nucleic acid molecules can be "partial," in which only some of the nucleotides bind, or it can be complete, in which total complementarity exists between the single-stranded molecules. The degree of complementarity between nucleic acid strands has a significant effect on the efficiency and strength of hybridization between nucleic acid strands.

[0132] "Selective hybridization" or "selective annealing" refers to the binding of a nucleic acid to a target nucleic acid without substantial binding to other nucleic acids present in a hybridization mixture under defined stringent conditions. Those skilled in the art recognize that relaxing the stringency of hybridization conditions allows for tolerance of sequence mismatches.

[0133] In some embodiments, hybridization occurs under stringent hybridization conditions. The phrase "stringent hybridization conditions" generally refers to conditions under which a particular sequence will hybridize to a perfectly matched sequence, but not to a sequence with mismatches. Typically, these conditions are in a medium that is at a defined ionic strength and pH, at a temperature that is about 5°C to about 20°C or 25°C below the melting temperature (T m ) of the particular sequence. As used herein, 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 T m 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, 2ND ED., VOLS. 1-3, Cold Spring Harbor Laboratory), the descriptions of which regarding stringent hybridization conditions are incorporated herein by reference. As indicated in the standard reference, T mA simple estimate of the value can be calculated by the following equation: T m = 81.5 + 0.41 (% G+C) (see, e.g., Quantitative Filter Hybridization in Anderson and Young, NUCLEIC ACID HYBRIDIZATION (1985)). The melting temperature of a hybrid (and thus the conditions for stringent hybridization) is influenced by various factors, such as the length and nature of the primer or probe (DNA, RNA, base composition) and the nature of the target nucleic acid (DNA, RNA, base composition, presence in solution or immobilized, etc.), as well as the concentration of salts and other components (e.g., presence or absence of formamide, dextran sulfate, polyethylene glycol). The effects of these factors are well known and discussed in standard references in the art. Illustrative stringent conditions suitable for most sequence-specific hybridizations are: a temperature of at least about 60°C and a salt concentration of about 0.2 moles / liter at pH 7. T m Calculations can be made 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, 1460-1465 (the description thereof regarding this is incorporated herein by reference).

[0134] The term “oligonucleotide” is used to refer to a relatively short nucleic acid, typically shorter than 200 nucleotides, more particularly shorter than 100 nucleotides, most particularly shorter than 50 nucleotides. Typically, an oligonucleotide is a single-stranded DNA molecule.

[0135] The term “primer” refers to an oligonucleotide that is capable of hybridizing (also referred to as “annealing”) to a nucleic acid and, under appropriate conditions (i.e., in the presence of the four different nucleotide triphosphates and reagents for polymerization, such as a DNA or RNA polymerase or reverse transcriptase), serves as a starting point for the polymerization of nucleotides (RNA or DNA) in an appropriate buffer at a suitable temperature. The appropriate length of a primer depends on the intended use of the primer, but a primer is typically at least 7 nucleotides long, and in some embodiments, is in the range of 10 to 30 nucleotides in length, or in some embodiments, is 10 to 60 nucleotides in length. In some embodiments, a primer can be, for example, 15 to 50 nucleotides long. Short primer molecules typically require lower temperatures in order to form sufficiently stable hybridization complexes with the template. A primer need not reflect the exact sequence of the template, but must be sufficiently complementary to hybridize with the template.

[0136] A primer is said to "anneal" or "hybridize" to another nucleic acid if the primer or a portion thereof hybridizes to a nucleotide sequence within the nucleic acid. The expression that a primer hybridizes to a particular nucleotide sequence is not intended to imply that the primer is completely hybridized or hybridized only to that nucleotide sequence. For example, in some embodiments, amplification primers used herein are referred to as "annealing" to or "specific for" a certain nucleotide sequence. The specification encompasses primers that anneal completely to a nucleotide sequence, as well as primers that anneal partially to a nucleotide sequence.

[0137] The term "primer pair" refers to a set of primers, including a 5' "upstream primer" or "forward primer" that hybridizes to the complementary strand of the 5' end of a DNA sequence to be amplified and a 3' "downstream primer" or "reverse primer" that hybridizes to the 3' end of the sequence to be amplified. As 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 directions in some embodiments.

[0138] A "probe" is a nucleic acid that is capable of binding to a target nucleic acid of complementary sequence through one or more types of chemical bonds, typically through complementary base pairing, usually through hydrogen bonding, to form a duplex structure. A probe can be labeled with a detectable moiety to allow easy detection of the probe, particularly after the probe has hybridized to its complementary target. Alternatively, however, a probe can be unlabeled, but detectable by specific binding to a labeled ligand, either directly or indirectly. Probes can vary greatly in size.

[0139] As used herein, the term "specific for a nucleic acid" with respect to a portion of a primer or a nucleotide sequence within a primer refers to a primer or nucleotide sequence that can specifically anneal to a target nucleic acid under suitable annealing conditions.

[0140] The term "target" is used herein to refer to both "target nucleic acids" as well as "target organisms". The former refers to nucleic acids to be detected, the latter to organisms to be detected. The term "target nucleic acid" is used herein generally to refer to a nucleic acid segment defined by a primer pair and which generates an amplicon in an amplification reaction; the term "amplification target" is also used herein to refer to this type of target nucleic acid. Primers and probes are also referred to as "targeting" a nucleic acid sequence, and thus these sequences can also be understood to be "target nucleic acids". In addition, primers and probes are said to be "specific for" a gene or "to have specificity for a gene". 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. Depending on the context in which the terms are used, the meaning of "target" and "target nucleic acid" will be clear to one of skill in the art. In some embodiments, multiple target nucleic acids can be detected to detect a single target organism. In some embodiments, a single target nucleic acid can be detected to detect a single target organism. In some embodiments, an assay can employ multiple target nucleic acids for one or more target organisms, and can also employ a single target nucleic acid for one or more different target organisms.

[0141] Amplification, in accordance with the teachings herein, encompasses any method that generally replicates at least a portion of at least one target nucleic acid in a template-dependent manner, including but not limited to a wide range of techniques for linear or exponential amplification of nucleic acid sequences. Illustrative methods for performing the amplification step include PCR, nucleic acid strand based amplification (NASBA), two-step multiplex amplification, rolling circle amplification (RCA), and the like, including multiplexed versions and combinations thereof, such as but not limited to OLA / PCR, PCR / OLA, LDR / PCR, PCR / PCR / LDR, PCR / LDR, LCR / PCR, PCR / LCR (also known as combined chain reaction - CCR), helicase-dependent amplification (HDA), 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.; U.S. Patent No. 6,027,998; U.S. Patent No. 6,605,451; Barany et al., PCT Publication No. WO 97 / 31256; Wenz et al., PCT Publication No. WO 01 / 92579; Day et al., Genomics, 29(1): 152-162 (1995); Ehrlich et al., 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); 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: e40 i-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(1): 21-7; U.S. Patent No. 5,830,711; U.S. Patent No. 6,027,889; U.S. Patent No. 5,686,243; PCT Publication No. WO0056927A3; and PCT Publication No. WO9803673A1.

[0142] In some embodiments, amplification comprises at least one cycle of the following steps in order: annealing at least one primer to a complementary or substantially complementary sequence in at least one target nucleic acid; using a polymerase to synthesize at least one strand of nucleotides in a template-dependent manner; and denaturing the newly formed nucleic acid duplex to separate the strands. The cycle can or can not be repeated. Amplification can comprise thermal cycling or can be performed isothermally.

[0143] As used herein, the term "amplification conditions" refers to conditions that facilitate amplification of a target nucleic acid in the presence of suitable primers.

[0144] As used herein, "in solution" means not immobilized on any kind of substrate, e.g., a surface in a microbead or cartridge, such as a chamber wall.

[0145] A "multiplex amplification reaction" is a reaction that simultaneously amplifies two or more nucleic acids that are distinguishable by sequence.

[0146] The term "qPCR" is used herein to designate 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.

[0147] The term "melting curve analysis" refers to the use of the dissociation characteristics of a double-stranded nucleic acid fragment during heating. Initially, UV absorbance measurements were used to observe strand dissociation, but now techniques based on fluorescence measurements are the most common approach. The temperature-dependent dissociation between two DNA strands can be measured in a "melting assay", for example using a DNA-intercalating fluorophore such as SYBR green or EvaGreen, or a fluorophore-labeled DNA probe. Using SYBR green as an example (which enhances fluorescence intensity 1000-fold when intercalated into the minor groove of both strands of DNA), the dissociation of DNA during heating can be measured by the large decrease in fluorescence produced. Alternatively, a TaqMan® probe (one characterized by a fluorophore and the other by a suitable quencher) can be used to determine the complementarity of the probe to the target nucleic acid sequence.

[0148] “Reagent” broadly refers to any reagent used in a reaction other than the analyte (e.g., nucleic acid being analyzed). Illustrative reagents for nucleic acid amplification reactions include, but are not limited to, buffers, metal ions, polymerases, reverse transcriptases, primers, template nucleic acids, nucleotides, labels, dyes, nucleases, dNTPs, and the like. Reagents for enzymatic reactions include, for example, substrates, cofactors, buffers, metal ions, inhibitors, and activators.

[0149] As used herein, the term “label” refers to any atom or molecule that can be used to provide a detectable and / or quantifiable signal. In particular, a label can be directly or indirectly attached to a nucleic acid or protein. Suitable labels that can be attached to a probe include, but are not limited to, radioisotopes, fluorophores, chromophores, mass labels, electron-dense particles, magnetic particles, spin labels, chemiluminescent molecules, electrochemically active molecules, enzymes, cofactors, and enzyme substrates.

[0150] As used herein, the term “dye” generally refers to any organic or inorganic molecule that absorbs electromagnetic radiation and produces a detectable signal (e.g., a fluorescent signal).

[0151] As used herein, the term “quencher” generally refers to any organic or inorganic molecule that reduces the level of a detectable signal.

[0152] As used herein, the term “detect” means “determine the presence of an item,” such as a nucleic acid sequence, e.g., a nucleic acid sequence indicative of the presence of a coronavirus. Detection can include determining the presence of a coronavirus without explicitly identifying that coronavirus; determining the presence of one or more coronaviruses belonging to a class of coronaviruses; determining the presence of a particular, known strain of coronavirus; or determining the presence of a novel (previously undescribed) strain of coronavirus.

[0153] As used herein, the term “treatment regimen” refers to any medical intervention aimed at alleviating the symptoms and / or pathology of a disorder. A treatment regimen can include one or more actions (e.g., bed rest, increased fluid intake), the use of a non-prescription or prescription drug, a supplement, a food, a drink, or a medical device (e.g., a ventilator).

[0154] As used herein, “Clinical Laboratory Improvement Amendments (CLIA)” refers to the 1988 Clinical Laboratory Improvement Amendments (CLIA) regulations, which were in effect as of the original filing date of this application. The CLIA regulations include federal standards applicable to all U.S. agencies or facilities that test human specimens for health assessments or to diagnose, prevent, or treat disease. A “CLIA compliant” test is one that complies with these regulations. A “CLIA exempt” test includes one that does not comply with all of these regulations. For example, CLIA exempt tests include test systems approved by the U.S. Food and Drug Administration for home use and those approved exempt from CLIA standards.

[0155] As used herein, the term “virulence” can refer to the degree of infectivity and / or the severity of disease induced by a pathogen, such as a virus. A virus that is “more virulent” is more infectious and / or induces a more severe disease compared to a reference virus, and vice versa. In many embodiments, a “avirulent” virus does not typically induce a disease that requires hospitalization.

[0156] As used herein, an “endogenous control” refers to a moiety that is naturally present in a sample to be used for detection. In some embodiments, the endogenous control is a “sample adequacy control” (SAC), which can be used to determine whether a sample used in an assay is adequate, or whether the sample contains sufficient biological material, such as cells. In some embodiments, the endogenous control is an RNA (such as mRNA, tRNA, ribosomal RNA, etc.), such as human RNA for a human sample. Non-limiting exemplary endogenous controls include ABL mRNA, GUSB mRNA, GAPDH mRNA, TUBB mRNA, and UPKla mRNA. In some embodiments, an endogenous control, such as a SAC, is selected that can be detected in the same manner as a target nucleic acid (e.g., RNA), and in some embodiments, is detected at the same time as the target nucleic acid (e.g., RNA).

[0157] As used herein, an “exogenous control” refers to a moiety added to a sample or assay, such as a “sample processing control” (SPC). In some embodiments, the exogenous control is included with the assay reagents. The exogenous control is typically selected to be expected to be absent from the sample to be used for detection, or to be present in the sample at a very low level, such that the amount of the moiety naturally present in the sample is not detectable, or is detectable at a level much lower than the amount added to the sample as an exogenous control. In some embodiments, the exogenous control comprises a nucleotide sequence that is expected to be absent from the sample type used to detect the target nucleic acid (e.g., RNA). In some embodiments, the exogenous control comprises a nucleotide sequence that is known to be absent from the species from which the sample is taken. In some embodiments, the exogenous control comprises a nucleotide sequence from a species different from the subject from which the sample is taken. In some embodiments, the exogenous control comprises a nucleotide sequence that is known to be absent from any species. In some embodiments, the exogenous control is selected that can be detected in the same manner as the target nucleic acid (e.g., RNA), and in some embodiments, is detected at the same time as the target nucleic acid (e.g., RNA). In some embodiments, the exogenous control is RNA. In some such embodiments, the exogenous control is an Armored which comprises RNA encased in a phage protective coating. See, e.g., Walker Peach et al., Clin. Chem. 45: 12: 2079-2085 (1999).

[0158] Respiratory Pathogens

[0159] It is an object of the present disclosure to identify selected respiratory pathogens by nucleic acid amplification-based methods. Such selections include alpha-coronavirus, beta-coronavirus, SARS-CoV-2, adenovirus, Chlamydia pneumoniae, influenza A, influenza B, metapneumovirus, rhinovirus / enterovirus, mycoplasma, bordetella, parainfluenza, and respiratory syncytial virus (RSV).

[0160] Coronavirus

[0161] Coronaviruses are enveloped, positive-sense ssRNA viruses in the family Coronaviridae. The subfamily Coronavirinae contains four genera: alphacoronavirus, betacoronavirus, gammacoronavirus, and deltacoronavirus. Alphacoronavirus and betacoronavirus infect mammals, including humans. Their virions contain four major structural proteins: nucleocapsid (N) protein, transmembrane (M) protein, envelope (E) protein, and spike (S) protein. In addition, the CoV genome encodes 16 non-structural proteins (nspl-16) that drive genome expression and replication. The CoV genome includes overlapping open reading frames la and lb, which express the large polyprotein ORFla and ORFlab, known as the replicase for its established role in viral RNA synthesis. ORFla or ORFlab is subsequently cleaved by viral proteases.

[0162] Seasonal coronaviruses include HKU1 and OC43 (betacoronavirus), as well as NL63 and 229E (alphacoronavirus), which cause mild to moderate upper respiratory tract infections. They are responsible for causing common cold episodes in humans worldwide. Infection is characterized by nasal discharge, nasal congestion, sore throat, sneezing, and cough with mild fever. Infection can also cause lower respiratory tract infections, including bronchitis and pneumonia, but this is not common.

[0163] The SARS-CoV-2 genome includes genes that encode four structural proteins (S, E, M, and N) and sixteen non-structural proteins (nspl-16). The nucleocapsid protein (N) forms a capsid outside the genome, which is further encased by an envelope associated with three structural proteins: the membrane protein (M), the spike protein (S), and the envelope protein (E). Nspl mediates RNA processing and replication. Among the non-structural proteins, Nsp 12 contains the RNA-dependent RNA polymerase (RdRp), which is a key component of coronavirus replication / transcription.

[0164] The N2 assay is specific for SARS-CoV-2; it does not detect other closely related coronaviruses in the Sarbecovirus subgenus, such as SARS-CoV-1. This assay was chosen to provide this high level of specificity for SARS-CoV-2. The E assay has “Sarbecovirus specificity” in that it detects not only SARS-CoV-2, but also other coronaviruses in the Sarbecovirus subgenus. Since SARS-CoV-2 is the only member of the Sarbecovirus subgenus known to currently be circulating in humans, it is effectively specific for SARS-CoV-2. However, if another Sarbecovirus emerged in the human population, the E assay would be fully capable of detecting it. In some embodiments, the assays described herein provide a high level of specificity for SARS-CoV-2 by targeting the N2, RdRp, and E genes. MERS-CoV is a member of the Betacoronavirus C lineage and would not be detected by the N2, RdRp, and E targets for SARS-CoV-2.

[0165] Adenovirus (AdV)

[0166] Adenoviruses AdV have a spherical structure of 70-100 nm, with an outer shell composed of 252 capsomeres, including 240 hexons and 12 pentons, which make up a icosahedral viral capsid. The genome contains early-expressed El-E4 genes that encode replication proteins, the intermediate genes IX and IVa2, and the late region genes LI-L5 that are associated with structural protein production. Currently, more than 84 genotypes have been identified, including all previously characterized serotypes that have been identified, based on their immunochemical responses, nucleic acid characteristics, hexon and fiber protein characteristics, biological properties, and phylogenetic relationships, and grouped into seven different species (A-G). These viruses can cause respiratory tract infections (AdV species B, C, and E), as well as ocular infections (conjunctivitis, keratoconjunctivitis), enteric infections (gastroenteritis), nervous system infections (meningitis and encephalitis), and urinary tract infections (AdV species A, D, F, and G). The viruses can cause upper respiratory tract (URTI) flu-like symptoms; pharyngitis (sore throat), rhinitis (runny nose), cough, ear infections, red eye, and fever. Treatment is primarily directed at symptomatic treatment, with acetaminophen for fever. In severe adenovirus infections, cidofovir has been used to treat immunocompromised individuals.

[0167] Chlamydia pneumoniae (Chlamydophila)

[0168] Chlamydia pneumoniae is an important intracellular pathogen that causes infection in more than one million people each year. It is an uncommon cause of community-acquired pneumonia and bronchitis. The incubation period of C. pneumoniae infection is 3 to 4 weeks and exhibits a broad spectrum of clinical symptoms. Most patients exhibit cough, fever, malaise, headache, and can develop pharyngitis, laryngitis, rhinitis, or pneumonia. The ompA gene of C. pneumoniae is highly conserved within the species.

[0169] Influenza Virus

[0170] Influenza viruses belong to the family Orthomyxoviridae and are the major cause of severe respiratory illness worldwide. Influenza viruses are transmitted from infected persons to healthy persons mainly through respiratory droplets. The typical features of influenza infection are fever, chills, myalgia, headache, rhinorrhea, cough, and upper respiratory tract inflammation. After accurate and timely diagnosis, the patient can be treated appropriately. Antiviral therapy based on the use of viral protein inhibitors is most commonly used.

[0171] There are four types of influenza viruses: types A, B, C, and D. Influenza A viruses (IAVs) are the only influenza viruses known to cause influenza pandemics, while influenza A and B viruses (IAVs and IBVs) almost every winter cause seasonal disease epidemics (referred to as influenza season). In contrast, influenza C infections usually cause mild illness and are not thought to cause influenza epidemics in humans, and diagnostic testing for influenza C viruses is not routinely performed. Influenza D viruses mainly affect cattle and are not known to infect humans or cause human illness.

[0172] The genome of influenza A and B viruses is composed of eight single-stranded viral RNA (vRNA) segments. Each segment (named after the corresponding protein) encodes one viral protein, including the major surface glycoproteins hemagglutinin (HA) and neuraminidase (NA), nucleocapsid protein (NP), three subunits of the viral RNA-dependent RNA polymerase (RdRP) (PA, PA-X, PB1, PB2, PB1-F2), matrix proteins (M1, M2), and the non-structural proteins NS1 and NS2. Influenza A is divided into 16 HA subtypes and 9 NA subtypes according to the antigenicity of the surface glycoproteins.

[0173] Importantly, influenza viruses have the ability to rapidly acquire adaptive genetic mutations in a process known as antigenic drift, whereby they gradually evade the human immune response. Additionally, the segmented genome enables the genetic material of different IAV strains to undergo special rearrangements, resulting in new genetically reassorted virus strains, a process known as antigenic shift, which periodically leads to the emergence of virus strains with significantly altered characteristics that have the ability to infect humans and enhance pathogenicity during pandemic outbreaks. While influenza A viruses undergo both antigenic drift and shift, and are the only influenza viruses known to cause pandemics, influenza B viruses change only through the more gradual process of antigenic drift.

[0174] Human Metapneumovirus (hMPV)

[0175] Human metapneumovirus (hMPV) is a negative-sense ssRNA virus of the Pneumoviridae family. hMPV causes 5-7% of RTIs in children and about 3% of RTIs in all ages. Symptoms in patients include cough, rhinorrhea, wheezing, dyspnea, and fever. During respiratory virus season, hMPV, RSV, and influenza can circulate simultaneously, making diagnosis difficult without confirmatory testing.

[0176] The hMPV genome is approximately 13,000 nucleotides in length and consists of eight genes that encode nine proteins: nucleoprotein (N), phosphoprotein (P), matrix protein (M), fusion protein (F), matrix-2 proteins (M2-1 and M2-2), small hydrophobic (SH) protein, glycoprotein (G), and large (L) polymerase protein. The N, L, and P proteins form the viral replication complex. Although hMPV is similar to RSV in genome, hMPV has a different gene order than RSV and lacks the nonstructural proteins NS1 and NS2.

[0177] Rhinovirus and Enterovirus

[0178] Rhinoviruses (RVs) and enteroviruses (EVs) are members of the Picornaviridae family. RVs and EVs are small RNA viruses composed of a small capsid containing a single-stranded, positive-sense RNA genome. The 5' end of the viral RNA includes a non-translated region (NTR) that is well-defined and highly conserved among RVs and EVs, making it a common target for molecular diagnostic testing. The 5'-NTR of picornaviruses has a higher G+C content than the rest of the picornavirus genome, which is important for stability of secondary structure and adaptation to the environment. The 5'-NTR region is characterized by a high degree of sequence homology among picornavirus species.

[0179] RV has three species, A, B, and C, with over 100 serotypes. All RV serotypes of species A, B, and C can cause respiratory symptoms. EV has many human and non-human species. Human EV species include A, B, C, and D. EVs are associated with many different human diseases, although many of these diseases can begin with mild upper respiratory symptoms, one EV serotype, D68, is associated with a unique respiratory illness.

[0180] Rhinovirus

[0181] Unlike EVs, most RVs lose infectivity when exposed to weak acids, which explains why they generally cannot infect the intestinal tract or spread via the fecal-oral route. RVs are the most common cause of the common cold (upper respiratory tract infection (URTI)) in all age groups. Complications of URTIs include acute otitis media and sinusitis. RVs, particularly species C, can cause lower respiratory tract disease. Transmission of RVs occurs via direct inoculation to the nose or eye, or via respiratory droplets. The disease is usually characterized by watery nasal discharge, malaise, nasal congestion, pharyngitis, and cough. Fever is not common.

[0182] RVs can be identified in most respiratory sample types, including swabs or aspirates from the nose, throat, or nasopharynx, and sputum, tracheal aspirates, and bronchoalveolar lavage (BAL) when lower respiratory symptoms are present. RVs of species A and B can be readily cultured in human cell lines. Members of species C RV are usually non-culturable. RVs in cell culture can be identified by the characteristic cytopathic effect (CPE) and distinguished from EVs (which have similar CPE) by their acid lability. This is a cumbersome laboratory test that exposes an aliquot of the sample to an acidic solution, followed by a culturability test. There are no antigen-based tests to detect RVs, nor are there serological tests to detect immune responses as a diagnosis of acute infection. The 5' NTR of RVs is highly conserved among picornaviruses.

[0183] Enterovirus

[0184] As previously mentioned, human EVs have four species, A-D. Species A EVs consist of several coxsackievirus A types (e.g., coxsackievirus A10) and "numbered" enterovirus types (e.g., enterovirus A71). Species B EVs include several coxsackievirus B types, as well as numbered enterovirus types. Species C EVs include several coxsackievirus A types, poliovirus, and numbered enteroviruses. Finally, species D includes several numbered enteroviruses, including enterovirus D68, which is a pathogen of severe lower respiratory tract disease in infants. Following infection, initial EV replication occurs in the upper respiratory tract, after which the virus can evade the host immune response, spread, and infect organ systems such as the central nervous system and heart. EVs can be transmitted via the fecal-oral route and the respiratory route. Although most EV infections are asymptomatic, it is estimated that millions of symptomatic EV infections occur annually in the United States. Most EV infections cause only mild, non-specific illness, but infection can also lead to severe disease, especially in infants. EVs are the most common cause of aseptic meningitis, and they all cause rash illnesses, such as hand-foot-and-mouth disease and herpangina, pericarditis, pleuritic pain, myocarditis, acute hemorrhagic conjunctivitis, acute respiratory illness, encephalitis, poliomyelitis (natural polio has been largely eradicated), and acute flaccid myelitis. While some of these diseases can be preceded by a mild common cold syndrome, EV D68 is of concern due to its association with severe lower respiratory tract disease in infants.

[0185] Most EVs can be isolated and identified in cell culture. However, virus isolation is complex, laborious, and can take days to weeks to detect cytopathic effects. Since both EVs and RVs can be isolated from the upper respiratory tract, and cytopathic effects in cell culture can appear identical, a technically demanding acid-lability test (previously described) needs to be performed. Confirmatory cell culture cytopathic effect-specific reagents for EV serotypes are commercially available for only a relatively small number of EV serotypes. There are no antigen-based tests or serological methods to diagnose acute infection.

[0186] The 5'-NTR is conserved in EVs as well as RVs (and other small RNA viruses, as described above), which makes it difficult to utilize this region to distinguish between the two.

[0187] Mycoplasma pneumoniae

[0188] Mycoplasma pneumoniae is a cell wall-less bacterium that causes community-acquired atypical pneumonia. In the United States, an estimated 2 million cases of Mycoplasma pneumoniae infection occur each year. The most common type of illness caused by this bacterial infection is tracheobronchitis. The main symptoms of infection include fatigue and sore throat, fever, and productive cough. Attachment to the respiratory epithelium is the first step in the infection process. Mycoplasma pneumoniae adhesion to its host cell is a complex event that requires a terminal structure at one end of the bacterial cell, the attachment organelle, which mediates the bacterial-host interaction. Within the membrane of the attachment organelle are clustered adhesion proteins including P1 adhesin.

[0189] Bordetella pertussis and parapertussis

[0190] Bordetella pertussis and parapertussis are gram-negative bacteria known to cause highly contagious whooping cough. Patients infected usually have a mild cold for several weeks, which progresses to a rapid cough, accompanied by a high-pitched "whoop" sound. Parapertussis is known to cause a milder pertussis-like infection.

[0191] Human Parainfluenza Virus (HPIV)

[0192] Human parainfluenza virus (HPIV) is a negative-sense, single-stranded, enveloped RNA virus of the Paramyxoviridae family. There are four different types of HPIV: HPIV-1, HPIV-2, HPIV-3, and HPIV-4. In the United States, parainfluenza viruses affect more than 200,000 people each year. Human parainfluenza type 1 is most common in children (<5 years old). Both human parainfluenza types 1 and 2 cause upper and lower respiratory tract illness, as well as croup. Human parainfluenza type 3 is more often associated with bronchitis, bronchiolitis, and pneumonia. Human parainfluenza type 4 is less common, but can cause severe lung infections.

[0193] The genomes of HPIV 1, 2, and 3 are similar in size (15.5-15.7 kb), while the genome of HPIV4 is slightly larger, however their six genes are in the same order. There are two viral surface proteins: the hemagglutinin-neuraminidase (HN) protein, which mediates attachment to host cell membranes, and the fusion (F) protein, which mediates fusion of the viral envelope with the host cell membrane. The N protein encapsulates the genomic RNA, forming a highly stable nucleocapsid. The phosphoprotein (P) and large polymerase protein (L) are associated with the nucleocapsid, while the matrix protein (M) coats the inner surface of the envelope.

[0194] Respiratory Syncytial Virus (RSV)

[0195] RSV is a medium-sized (-150 nm) enveloped virus. The scientific name for this virus species is Human orthopneumovirus. It is synonymous with human respiratory syncytial virus (hRSV). It belongs to the Pneumoviridae family. Based on the reactivity of the F and G surface proteins to monoclonal antibodies, RSV is divided into two antigenic subtypes, A and B. Generally, RSV subtype A (RSVA) is considered more virulent than RSV subtype B (RSVB), with higher viral load and faster transmission. RSV infection can present with a wide range of signs and symptoms, ranging from mild upper respiratory tract infection (URTI) to severe and potentially life-threatening lower respiratory tract infection (LRTI) requiring hospitalization and mechanical ventilation. Treatments include antiviral therapy, anti-inflammatory drugs, and bronchodilators.

[0196] RSV is a negative-sense, single-stranded RNA virus. Its genome is linear, approximately 15000 nucleotides in length. [9] It is non-segmented, meaning, unlike influenza, RSV cannot participate in genetic reassortment and antigenic shift leading to large-scale epidemics. [4] It has 10 genes, encoding 11 proteins. [2] [4] The gene order is NS1-NS2-N-P-M-SH-G-F-M2-L, with the NS1 and NS2 genes serving as nonstructural promoter genes.

[0197] Respiratory Pathogen Biomarker Targets

[0198] In some embodiments, the respiratory pathogen discussed above can be detected by nucleic acid amplification in a respiratory panel assay, for example, in a multiplex amplification reaction that can be designed to detect 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 or more target nucleic acids per amplification reaction mixture. This degree of multiplexing can be achieved by using primers and probes that do not substantially cross-react or bind off-target and that can reliably identify the pathogen they target in the face of antigenic drift. Bioinformatic analysis of multiple databases can be performed to identify primers and probes to highly conserved regions of these pathogen genomes.

[0199] This article acknowledges the various limitations of currently available multiplex PCR methods. For example, while multiplexed amplification reactions for a large number of targets can be performed (e.g., multiplex PCR), simultaneous detection of multiple amplicones is not straightforward. To date, multiplex q-PCR methods, defined as the process of simultaneously amplifying and detecting multiple nucleic acid sequences in a single reaction chamber, have been implemented for a small number of amplicones, typically fewer than ten. Significant interest lies in efficiently performing multiplex assays within the same reaction volume and achieving simultaneous amplification and detection of multiple targets in the same reaction chamber. Such methods would not only better utilize the original DNA sample but also significantly reduce any of the complexities associated with the fluidic and liquid handling steps of running multiple single-reaction processes.

[0200] Attempts to establish multiplex q-PCR methods have been hampered by the practical problem of simultaneously detecting different nucleic acid sequences in a single sample. One possible approach is to associate different reporter molecules (e.g., fluorescent dyes) with a single amplicon during the PCR reaction, which allows for the parallel detection of a single reporter molecule by different “colors.” While such an approach theoretically provides parallelism, it is limited by: (i) the number of different reporter molecules available; (ii) crosstalk in the channels due to light signals in adjacent channels; and (iii) the availability of imagers and detectors capable of distinguishing different signals. Another possible approach to provide multiplexing capability is to separate the biological sample of interest and physically place it in separate, single, and isolated amplification chambers using a fluidic system. While this method can efficiently establish multiplex q-PCR by performing multiple singleton (i.e., one amplicon per chamber) q-PCR reactions, it may be suboptimal because when the original sample concentration is low, it may reduce the number of target nucleic acid sequences in each chamber, which may introduce random anomalies (Poisson noise) in the acquired data. Furthermore, it requires complex fluid handling steps.

[0201] High-level multiplexing of DNA sequences in a sample can be achieved using analytical platforms such as DNA microarrays or next-generation DNA sequencers, but not using q-PCR or equivalent techniques. Specifically, microarrays are large-scale parallel, affinity-based biosensors that selectively capture target nucleic acids from the same sample at different addressable coordinates (e.g., pixels) on a solid surface. Each addressable coordinate can have a unique DNA or RNA capture probe that is complementary to the target nucleic acid sequence to be detected in the sample. While microarrays offer high multiplexing capabilities, they are semi-quantitative and have poor limits of detection (LOD) and detection dynamic range (DDR) due to their endpoint detection nature (i.e., no real-time detection) and the fact that they lack any target amplification.

[0202] Because primers, probes, and target amplicones have a broad target range, a multiplex strategy is followed to screen and select primers and oligonucleotides. The multiplex design strategy for the Respiratory Panel may involve the following steps: single-base design of all primers / probes; multiplexing with background oligonucleotides and primer / probe matrix; multiplexing with primers and probes in separate pools; sequencing of the sample; redesigning primers / probes if necessary; multiplexing with all combined primers / probes together; and repeating one or more steps as needed. In this regard, numerous non-specific interactions caused by oligonucleotides were observed during assay development. Based on sequencing of PCR products and comprehensive computer simulation analysis, the oligonucleotides with the weakest interactions were selected. The assay was also optimized for the presence of salts, enzymes, oligonucleotide concentrations, and PCR procedures for more than 26 amplicones in solution.

[0203] In addition, to increase the number of target nucleic acids detected in each channel, the following method is used: (i) using T m (ii) Combining Taqman probes and melting probes in the same channel using melting probes below the annealing temperature (no amplification curve); and (ii) several melting target nucleic acids in one channel; the melting window for each target depends on the target's sequence variations. Several channel selections and designs were investigated for each target to find the optimal arrangement. As an example, the same probe was used in one channel to detect rhinovirus and enterovirus targets. The probes used had more mismatches with various enterovirus species (approximately 70% complementarity), resulting in detection by melting at low melting temperatures.

[0204] Because the mutation rate of viral target organisms is high, making it difficult to find conserved regions, amplification detection is preferred to avoid melting probes and mutation-related T cells. m Major changes to the window.

[0205] Since a large number of oligonucleotides combined in a reaction mixture can lead to unwanted interactions between them, in some embodiments, modified nucleotides can be used to reduce primer interactions.

[0206] Table A below provides reference genomes of the genes targeted in the illustrative respiratory biomarker ensemble, along with primers, probes, and their locations. Specific implementations of the respiratory biomarker ensemble may include one or more amplification targets for each organism being tested. For example, in various implementations, organisms 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or all organisms listed in Table A, may be detected using two, three, or more amplification targets for each organism.

[0207] Table A: Nucleic Acid Targets for Respiratory Biomarkers

[0208]

[0209] Some implementations of respiratory biomarker combinations can detect one or more targets from one or more additional viral or bacterial pathogens, such as novel coronavirus, Hantavirus, cytomegalovirus, Coxsackievirus, herpes simplex virus, Echovirus, influenza C virus, Streptococcus pneumoniae, Moraxella catarrhalis, Haemophilus influenzae, Haemophilus parainfluenzae, Group A Streptococcus, Streptococcus pyogenes, Klebsiella pneumoniae, Pseudomonas spp., Neisseria spp., Histoplasma capsulatum, Coccidioides immitis, Blastomyces dermatitidis, Paracoccidioides brasiliensis, Candida spp., Aspergillus spp., Mucor spp., Cryptococcus neoformans, or Pneumocystis carinii.

[0210] Non-limiting exemplary primer pairs and optional probes are shown in Table B.

[0211] Table B: Primer, probe, and amplicon nucleotide sequences for respiratory biomarker targets

[0212]

[0213]

[0214]

[0215]

[0216]

[0217]

[0218]

[0219] Considerations for primers and optional probes for detecting respiratory pathogen biomarkers are detailed in the section entitled “Exemplary Polynucleotides” below.

[0220] The illustrative respiratory biomarker combination assay using primers and probes listed in Table B comprises a total of 80 oligonucleotides. Of these, 53 are primers, of which 38 are “simple” primers, which may include 0, 1, 2, or 3 sterically hindered groups, and 15 are modified primers, containing one or more sterically hindered groups and one or more chemically modified bases. Such sterically hindered groups and chemically modified bases provide greater primer-target complex stability and barriers to erroneous amplification (such as reducing primer-dimer formation and blocking certain primer extensions) compared to unmodified primers. The assay also includes 27 probes, of which 22 are single-dye probes and 5 are dual-dye probes. More information on polynucleotide modifications is provided in the section titled “Polynucleotide Modifications” below. More information on dyes and other markers useful in this assay is provided in the section titled “Polynucleotide Markers” below.

[0221] Controls

[0222] In some embodiments, the assay described herein includes the detection of the aforementioned respiratory pathogen biomarkers and at least one endogenous control. In some embodiments, the endogenous control is a sample adequacy control (SAC). In some such embodiments, if neither the respiratory pathogen biomarker nor the SAC is detected in the sample, the assay result is considered "invalid" because the sample may be insufficient. While not wishing to be bound by any particular theory, an insufficient sample may be too diluted, contain too little cellular material, or contain assay inhibitors, etc. In some embodiments, the absence of SAC may indicate a failed assay response. In some embodiments, the endogenous control is RNA (such as mRNA, tRNA, ribosomal RNA, etc.). Non-limiting exemplary endogenous controls include ABL mRNA, GUSB mRNA, GAPDH mRNA, TUBB mRNA, and UPKla mRNA.

[0223] In some embodiments, the assay described herein includes the detection of the aforementioned respiratory pathogen biomarkers and at least one exogenous control. In some embodiments, the exogenous control is a sample processing control (SPC). In some such embodiments, if the aforementioned respiratory pathogen biomarkers are not detected in the sample and the SPC is also not detected in the sample, the assay result is considered "invalid" because errors may have occurred during sample processing, including but not limited to assay failure. Non-limiting exemplary errors in sample processing include: inadequate sample processing, presence of assay inhibitors, presence of nucleases (such as RNases), or impaired reagents. In some embodiments, an exogenous control (such as an SPC) is added to the sample. In some embodiments, an exogenous control (such as an SPC) is added during the assay, such as with one or more buffers or reagents. In some embodiments, when using When the system is running, SPC is included. In the box. In some implementations, the exogenous control (such as SPC) is Armored. It is protected by the bacteriophage shell.

[0224] In some embodiments, the detection of endogenous and / or exogenous controls is performed simultaneously with the detection of respiratory pathogen biomarkers, for example, in the same assay. In some embodiments, the assay includes reagents for simultaneously detecting the aforementioned respiratory pathogen biomarkers and SAC and / or exogenous controls in the same assay reaction mixture. In some such embodiments, the assay reaction mixture includes, for example, a primer set for amplifying the aforementioned respiratory pathogen biomarkers, a primer set for amplifying SAC, and / or a primer set for amplifying the exogenous control, and an optional labeled probe (such as, for example, a probe for detecting the amplification product) for detecting the amplification product. (Probe).

[0225] Polynucleotides

[0226] In some embodiments, a polynucleotide is provided for detecting the aforementioned biomarkers. In some embodiments, a synthetic polynucleotide is provided. As used herein, a synthetic polynucleotide refers to a polynucleotide synthesized in vitro chemically or enzymatically. Chemical synthesis of polynucleotides includes, but is not limited to, synthesis using a polynucleotide synthesizer such as OligoPilot. TM (GE Healthcare), ABI 3900 DNA synthesizer (Applied Biosystems), etc. Enzymatic synthesis includes, but is not limited to, the production of polynucleotides through enzymatic amplification, such as PCR. Polynucleotides may contain one or more analogs of canonical nucleotides (e.g., modified nucleotides).

[0227] In some embodiments, a polynucleotide is provided comprising at least 6, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, or at least 2 [unclear - possibly related to respiratory pathogen targets and / or the exemplary controls discussed above]. Eight, at least 29, or at least 30 consecutive nucleotides that are 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% identical, or regions that are 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.

[0228] In various embodiments, the exemplary polynucleotide comprises at least: 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides. In various embodiments, the polynucleotide comprises fewer than: 200, 150, 100, 50, 40, 30, or 20 nucleotides. In various embodiments, the exemplary polynucleotide has a length of 6 to 200, 8 to 200, 8 to 150, 8 to 100, 8 to 75, 8 to 50, 8 to 40, 8 to 30, 15 to 100, 15 to 75, 15 to 50, 15 to 40, or 15 to 30 nucleotides.

[0229] In some embodiments, detection of each target nucleic acid can be performed using primers or probes with a single label specific to each target nucleic acid. Different primers and / or probes can have the same label. Multiple target nucleic acids can be detected simultaneously in a single reaction mixture by using primers or probes labeled with different detectable moieties (e.g., different fluorescent reporter dyes). In some embodiments, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more different labels can be used in a single reaction mixture or multiple reaction mixtures. Each target nucleic acid can be monitored independently using such multiplexing techniques. In some embodiments, detection of multiple target nucleic acids can be performed using primers or probes with a single label. Melting curves can be generated to distinguish two or more target nucleic acids each using the same label, but such analysis may not be necessary.

[0230] Polynucleotide Modifications

[0231] In some embodiments, the methods described herein for detecting at least one target nucleic acid employ one or more modified polynucleotides, such as polynucleotides containing one or more affinity-enhancing nucleotide analogs. Modified polynucleotides useful in the methods described herein include primers for reverse transcription, PCR amplification primers, and probes. In some embodiments, incorporation of affinity-enhancing nucleotides increases the binding affinity and specificity of the polynucleotide to its target nucleic acid compared to polynucleotides containing only canonical deoxynucleotides, allowing the use of shorter polynucleotides or shorter complementary regions between the polynucleotide and the target nucleic acid.

[0232] In some embodiments, the affinity-enhancing nucleotide analogues comprise nucleotides containing one or more base modifications, sugar modifications, and / or backbone modifications. In some embodiments, the modified bases for the affinity-enhancing nucleotide analogues include 5-methylcytosine, isocytosine, pseudoisocytosine, 5-bromouracil, 5-propynyluracil, 6-aminopurine, 2-aminopurine, inosine, diaminopurine, 2-chloro-6-aminopurine, xanthine, and hypoxanthine. In some embodiments, the affinity-enhancing nucleotide analogues comprise nucleotides with modified sugars, such as 2'-substituted sugars, such as 2'-O-alkyl-ribose, 2'-amino-deoxyribose, 2'-fluoro-deoxyribose, 2'-fluoro-arabinose, and 2'-O-methoxyethyl-ribose (2'MOE) sugars. In some embodiments, the modified sugar is arabinose or d-arabino-hexitol sugar.

[0233] In some embodiments, affinity-enhancing nucleotide analogs include backbone modifications, such as the use of peptide nucleic acids (PNAs; for example, oligomers comprising nucleobases linked together by an amino acid backbone). Other backbone modifications include phosphate thioester bonds, phosphodiester-modified nucleic acids, combinations of phosphodiester and phosphate thioester nucleic acids, methylphosphonates, alkylphosphonates, phosphate esters, alkyl phosphate thioesters, aminophosphate esters, carbamates, carbonates, triphosphate esters, acetamidates, carboxymethyl esters, methyl phosphate thioesters, dithiophosphate esters, p-ethoxy modifications, and combinations thereof.

[0234] In some embodiments, the polynucleotide includes at least one affinity-enhancing nucleotide analog having a modified base, a nucleotide having at least a modified sugar (which may be the same nucleotide), and / or at least one non-naturally occurring internucleotide bond.

[0235] In some embodiments, the affinity-enhancing nucleotide analog contains a locked nucleic acid (“LNA”) sugar, which is a bicyclic sugar. In some embodiments, the polynucleotide used in the methods described herein comprises one or more nucleotides having an LNA sugar. In some embodiments, the polynucleotide contains one or more regions consisting of nucleotides having an LNA sugar. In other embodiments, the polynucleotide contains nucleotides having an LNA sugar, with the nucleotides having an LNA sugar alternating with deoxynucleotides. See, for example, Frieden, M. et al. (2008) Curr. Pharm. Des. 14(11):1138-1142.

[0236] Primers

[0237] In some implementations, the polynucleotide is the primer. Primers useful in the methods described herein are typically capable of selectively hybridizing to: genomic DNA, target RNA (genomic or transcript), cDNA reverse transcribed from the target RNA, and / or amplicons already amplified from genomic DNA, target RNA, or cDNA (collectively, the “template”), and in the presence of the template, polymerase, and suitable buffers and reagents, the primer can be extended to form a primer extension product. Primers are typically of sufficient length to ensure selective hybridization with their target nucleic acid. Typically, primers of at least 15 nucleotides in length hybridize specifically in most cases, and this length can be shortened, for example, by including affinity-enhancing modifications, such as those discussed above. Primers can, but do not necessarily, be perfectly complementary to their target nucleic acid. Primers can have any degree of complementarity described above for the exemplary polynucleotide. In illustrative embodiments, primers can be 8 to 40 nucleotides long and at least 90% complementary to their target nucleic acid; 8 to 40 nucleotides long and at least 95% complementary to their target nucleic acid; 8 to 40 nucleotides long and at least 99% complementary to their target nucleic acid; 8 to 30 nucleotides long and at least 90% complementary to their target nucleic acid; 8 to 30 nucleotides long and at least 95% complementary to their target nucleic acid; 8 to 30 nucleotides long and at least 99% complementary to their target nucleic acid. In embodiments where the primer is less than 100% complementary to its target nucleic acid, the presence of a 3' nucleotide complementary to the target nucleic acid in the primer facilitates the generation of the extension product.

[0238] In some embodiments, primers that selectively hybridize with the target nucleic acid hybridize with its target nucleic acid under the same assay conditions with an affinity at least 5 times higher than that with non-target nucleic acids. In some embodiments, primers that selectively hybridize with the target nucleic acid hybridize with its target nucleic acid under the same assay conditions with an affinity at least 10 times higher than that with non-target nucleic acids.

[0239] In some implementations, primer pairs are designed to generate amplicons of 50 to 1500 nucleotides, 50 to 1000 nucleotides, 50 to 750 nucleotides, 50 to 500 nucleotides, 50 to 400 nucleotides, 50 to 300 nucleotides, 50 to 200 nucleotides, 50 to 150 nucleotides, 100 to 300 nucleotides, 100 to 200 nucleotides, or 100 to 150 nucleotides.

[0240] In some implementations, the primers are labeled with a detectable portion. In some implementations, the primers are not labeled.

[0241] Probes

[0242] In some implementations, the polynucleotide is the probe. Probes useful in the methods described herein are typically capable of selectively hybridizing to: genomic DNA, target RNA (genomic or transcript), cDNA reverse-transcribed from the target RNA, and / or amplicons already amplified from genomic DNA, target RNA, or cDNA (collectively, the “template”). Typically, probes of at least 15 nucleotides in length hybridize specifically in most cases, and this length can be shortened, for example, by including affinity-enhancing modifications, such as those discussed above. The probe can, but does not necessarily, be perfectly complementary to its target nucleic acid. For example, the probe can intentionally include a “mismatch” to modulate the Tm of the melting probe. The probe can have any degree of complementarity described above for the exemplary polynucleotides. In illustrative embodiments, the probe can be 8 to 40 nucleotides long and at least 70% complementary to its target nucleic acid; 8 to 40 nucleotides long and at least 75% complementary to its target nucleic acid; 8 to 40 nucleotides long and at least 80% complementary to its target nucleic acid; 8 to 40 nucleotides long and at least 85% complementary to its target nucleic acid; 8 to 40 nucleotides long and at least 90% complementary to its target nucleic acid; 8 to 40 nucleotides long and at least 95% complementary to its target nucleic acid; 8 to 40 nucleotides long and at least 99% complementary to its target nucleic acid; 8 to 30 nucleotides long and at least 90% complementary to its target nucleic acid; 8 to 30 nucleotides long and at least 95% complementary to its target nucleic acid; 8 to 30 nucleotides long and at least 99% complementary to its target nucleic acid. In embodiments where the probe is less than 100% complementary to the target nucleic acid, any non-complementary points or regions are typically positioned such that the probe's ability to selectively hybridize with its target nucleic acid is not impaired.

[0243] In some embodiments, the probe selectively hybridizes with the target nucleic acid and hybridizes with it under the same assay conditions with an affinity at least 5 times higher than that with non-target nucleic acids. In some embodiments, the probe selectively hybridizes with the target nucleic acid and hybridizes with it under the same assay conditions with an affinity at least 10 times higher than that with non-target nucleic acids.

[0244] Polynucleotide Labels

[0245] In some embodiments, primers or probes are labeled with a detectable portion. The detectable portion includes a directly detectable portion, such as a fluorescent dye, and an indirectly detectable portion, such as a binding pair member. When the detectable portion is a binding pair member, in some embodiments, the probe can be made detectable by incubating it with a detectable label bound to a second member of the binding pair. In some embodiments, primers or probes are unlabeled, such as when they are immobilized on, for example, microarrays or microbeads. Labeled primers are extendable, for example, by polymerase. In some embodiments, probes are extendable. In other embodiments, probes are non-extendable. The following discussion focuses on probes, as these probes are more commonly used for detection in the methods described herein, but those skilled in the art will understand that the polynucleotide labeling strategies described below are equally applicable to primer labeling.

[0246] In some embodiments, the probe is a FRET probe, and in some embodiments, its 5' end is labeled with a fluorescent dye (donor) and its 3' end with a quencher (acceptor). The quencher is a chemical group that absorbs (i.e., suppresses) the fluorescence emitted by the dye when the group is very close (e.g., attached to the same probe). Therefore, in some embodiments, the emission spectrum of the dye should have a large overlap with the absorption spectrum of the quencher. In other embodiments, the dye and quencher are not at the end of the FRET probe.

[0247] Explanatory FRET probes include, but are not limited to, Probes, molecular beacon probes, and scorpion probes. The probe is a linear probe, typically having a fluorescent dye covalently bound to one end of DNA and a quencher molecule covalently bound to other sites, such as the other end of the DNA. FRET probes contain sequences complementary to regions of cDNA or amplicons, such that when the FRET probe hybridizes with cDNA or amplicons, the dye fluorescence increases due to the increased distance between the dye and the quencher; when the FRET probe does not hybridize, the dye fluorescence is quenched; and when the probe is cleaved by an enzyme during the amplification of cDNA or amplicons, the dye is released from the probe and produces a fluorescent signal. In some embodiments, the amount of target nucleic acid in the sample is proportional to the amount of fluorescence measured during amplification.

[0248] and Similar to probes, molecular beacons use FRET to detect PCR products via probes, which have a fluorescent dye and a quencher attached to their ends. Unlike probes, molecular beacons remain intact during PCR cycles. When molecular beacon probes are free in solution, they form stem-loop structures, allowing the dye and quencher to come close enough to induce fluorescence quenching. When the molecular beacon hybridizes with the target nucleic acid, the stem-loop structure is disrupted, causing the dye and quencher to spatially separate, and the dye emits fluorescence. Molecular beacons can be obtained from, for example, GeneLink. TM Obtain (see www.genelink.com / newsite / products / mbintro.asp).

[0249] In some implementations, scorpion probes can be used as sequence-specific primers for PCR product detection. Like molecular beacons, scorpion probes form a stem-loop structure when not hybridizing with the target nucleic acid. However, unlike molecular beacons, scorpion probes achieve both sequence-specific priming and PCR product detection. A fluorescent dye molecule is attached to the 5' end of the scorpion probe, and a quencher is attached to other sites, such as the 3' end. The 3' portion of the probe is complementary to the extension product of the PCR primer, and this complementary portion is linked to the 5' end of the probe via a non-amplifiable portion. Upon extension of the scorpion primer, the target-specific sequence of the probe binds to the complementary strand within the extended amplicon, thereby opening the stem-loop structure, causing the dye at the 5' end to fluoresce and generate a signal. Scorpion probes are available from, for example, Premier Biosoft International (see www.premierbiosoft.com / tech_notes / Scorpion.html).

[0250] In some implementations, labels that can be used on FRET probes include colorimetric dyes and fluorescent dyes, such as Alexa Fluor dyes; BODIPY dyes, such as BODIPY FL, Cascade Blue, and Cascade Yellow; coumarins and their derivatives, such as 7-amino-4-methylcoumarin, aminocoumarin, and hydroxycoumarin; anthocyanin dyes, such as Cy3 and Cy5; eosin and erythrosine; fluorescein and its derivatives, such as fluorescein isothiocyanate; and macrocyclic chelates of lanthanides, such as Quantum Dye. TM Marina Blue; Oregon Green; Rhodamine dyes, such as Rhodamine Red, Tetramethylrhodamine and Rhodamine 6G; Texas Red; Fluorescent energy transfer dyes, such as Thiazole Orange-Ethidium Heteromer; and TOTAB.

[0251] 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 493 / 503, BODIPY 530 / 550, BODIPY 558 / 568, BODIPY 564 / 570, BODIPY 576 / 589, BODIPY 593 / 505, BODIPY 595 / 568, BODIPY 594 ... 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'-tetrabromosulfonylfluorescein and TET.

[0252] Examples of dye / quencher pairs (i.e., donor / acceptor pairs) include, but are not limited to, fluorescein / tetramethylrhodamine; IAEDANS / fluorescein; EDANS / dabcyl; fluorescein / fluorescein; BODIPY FL / BODIPY FL; and fluorescein / QSY7 or QSY 9 dyes. In some embodiments, when the donor and acceptor are the same, FRET can be detected by fluorescence depolarization. Specific examples of dye / quencher pairs (i.e., donor / acceptor pairs) include, but are not limited to, Alexa Fluor 350 / Alexa Fluor 488; 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 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 QSY 9; 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 cases, the same quencher can be used for multiple dyes, such as broad-spectrum quenchers like Iowa. Quencher (Integrated DNA Technologies, Coralville, IA) or Black Hole Quencher TM (BHQ TM ; Sigma-Aldrich, St. Louis, MO).

[0253] Specific examples of fluorescently labeled ribonucleotides useful in preparing probes for some embodiments of the methods described herein are available from Molecular Probes (Invitrogen), including: Alexa Fluor 488-5-UTP, Fluorescein-12-UTP, BODIPY FL-14-UTP, BODIPY TMR-14-UTP, Tetramethylrhodamine-6-UTP, AlexaFluor 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.

[0254] Specific examples of fluorescently labeled deoxyribonucleotides useful in preparing probes for the methods described herein include dinitrophenyl (DNP)-1'-dUTP, Cascade Blue-7-dUTP, Alexa Fluor 488-5-dUTP, fluorescein-12-dUTP, Oregon Green 488-5-dUTP, BODIPY FL-14-dUTP, Rhodamine Green-5-dUTP, Alexa Fluor 532-5-dUTP, BODIPY TMR-14-dUTP, tetramethylrhodamine-6-dUTP, Alexa Fluor 546-14-dUTP, Alexa Fluor 568-5-dUTP, Texas Red-12-dUTP, Texas Red-5-dUTP, BODIPY TR-14-dUTP, Alexa Fluor 594-5-dUTP, and BODIPY... 630 / 650-14-dUTP, BODIPY 650 / 665-14-dUTP; Alexa Fluor 488-7-OBEA-dCTP, Alexa Fluor 546-16-OBEA-dCTP, Alexa Fluor 594-7-OBEA-dCTP, and Alexa Fluor 647-12-OBEA-dCTP. The fluorescently labeled nucleotides are commercially available and can be purchased from, for example, Invitrogen.

[0255] As described above, the exemplary detectable portion also includes binding pair members. Exemplary binding pairs include, but are not limited to, biotin and streptavidin, antibodies and antigens.

[0256] Samples

[0257] The sample to be tested can be any sample suspected of containing at least one of the respiratory pathogen biomarkers described herein. In some embodiments, the sample is a biological sample collected from a subject. In other embodiments, the sample is a sample not collected directly from a subject, such as a wastewater sample or a sample from an air filter within a building.

[0258] Explanatory biological samples include body fluid samples, such as nasal aspirates, nasal washes, nasal swabs, nasopharyngeal swabs, saliva, oropharyngeal swabs, pharyngeal swabs, bronchoalveolar lavage fluid samples, bronchial aspirates, bronchial lavage fluid, tracheal aspirates, tracheal lavage fluid, tracheal aspirates, nasal secretions, mucus samples, sputum samples, lung tissue samples, etc.

[0259] In some embodiments, the sample to be tested is fresh (i.e., never frozen). In other embodiments, the sample is a frozen sample. In some embodiments, the sample is a tissue sample, such as a formalin-fixed paraffin-embedded sample. In some embodiments, the sample is a liquid cytology sample.

[0260] In some embodiments, the sample to be tested is contacted with a buffer solution 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 solution (including, for example, a preservative) may be added to the nasal aspirate sample or nasal wash sample. In embodiments where the sample is a nasopharyngeal swab sample, the swab may simply be placed in the buffer solution. In some embodiments, the sample is contacted with the buffer solution immediately; for example, a swab is placed in the buffer solution immediately. In some embodiments, the sample (e.g., including a swab) is contacted with the buffer solution within 5 minutes, 10 minutes, 30 minutes, 1 hour, or 2 hours after sample collection.

[0261] In some embodiments, this method uses samples smaller than 5 ml, 4 ml, 3 ml, 2 ml, 1 ml, or 0.75 ml, or samples with added buffer. In some embodiments, this method uses samples from 0.1 ml to 1 ml, or samples with added buffer.

[0262] Subjects

[0263] In the methods described herein, useful biological samples can be collected from any subject who may be infected with one, several, or all of the aforementioned respiratory pathogens. In various embodiments, the subject may include non-human animals, such as canines, felines, equines, primates, and other non-human mammals, as well as humans.

[0264] In some embodiments, the test sample is obtained from an individual exhibiting one or more symptoms of influenza infection. Non-limiting exemplary symptoms of influenza include fever, chills, cough, sore throat, runny nose, nasal congestion, muscle aches, headache, fatigue, vomiting, diarrhea, and any combination of these symptoms. In some embodiments, the test sample is obtained from an individual previously diagnosed with an illness caused by a respiratory pathogen (e.g., influenza or Covid-19). In some such embodiments, recurrence of illness caused by a respiratory pathogen (e.g., influenza or Covid-19) in the individual is monitored.

[0265] In some embodiments, the methods described herein can be used for routine screening of seemingly healthy individuals without risk factors. In some embodiments, the methods described herein are used to screen asymptomatic individuals, such as during routine or preventative healthcare. In some embodiments, the methods described herein are used to screen pregnant women or women planning to conceive.

[0266] In some implementations, the methods described herein can be used to assess the effectiveness of treatment for individuals undergoing treatment for symptoms caused by respiratory pathogens (e.g., influenza or Covid-19).

[0267] Assay Methods

[0268] Any analytical step that allows for the specific detection of the target nucleic acid can be used in the methods proposed herein. In some embodiments, DNA targets can be detected by direct hybridization, or more readily, by amplification of the DNA template and detection of the amplicon. In some embodiments, RNA targets can be detected by direct hybridization, or more readily, by reverse transcription of the target RNA to generate cDNA complementary to the target RNA. This cDNA can be detected directly by direct hybridization or by amplification of the cDNA template.

[0269] Nucleic acid amplification provides rapid, sensitive, and specific detection of nucleic acid targets and has been used in a variety of assays. Those skilled in the art can follow the guidance herein and use any of the nucleic acid amplification methods discussed above to perform the methods described herein in a variety of different nucleic acid amplification-based assays. Such methods may require thermal cycling, but are not necessarily necessary, as exemplified by isothermal amplification. Exemplary methods include, but are not limited to, isothermal amplification, real-time RT-PCR, endpoint RT-PCR, and amplification from annealed DNA to the T7 promoter using T7 polymerase, such as SenseAmp Plus, available in Implen, Germany. TMThe kit is provided with the reagent kit. Amplification and detection can be performed in solution or using 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.

[0270] In some embodiments, target nucleic acids, such as respiratory and other pathogen biomarkers and / or optional controls, can be detected by the following steps: (a) contacting nucleic acids from a sample with a set of primers and optional probes for detecting the presence of the target nucleic acid; (b) placing the nucleic acid, primers, and optional probes under amplification conditions; (c) detecting the presence of any (multiple) amplification products, optionally via real-time PCR, melting curve analysis, or a combination thereof; and (d) differentially identifying the presence of viral and / or bacterial respiratory pathogens in the sample, or determining the absence of viral or bacterial pathogens detectable using the primer set, based on the detection or absence of the (multiple) amplification products. In this context, "differential identification" refers to the ability to determine the presence of a specific target organism while the absence of one or more other target organisms is measured. In some embodiments, the assay is capable of determining the presence of any target organism present in the sample while excluding the presence of other target organisms (above the detection limit of the assay).

[0271] In some embodiments of amplification via polymerase chain reaction (PCR), exemplary cycles include initial denaturation at 90°C to 100°C for 20 seconds to 5 minutes, followed by cycles of denaturation at 90°C to 100°C for 1 to 10 seconds, followed by annealing and amplification at 60°C to 75°C for 10 to 40 seconds. Another exemplary cycle includes 20 seconds at 94°C, followed by up to 3 cycles of 1 second at 95°C and 35 seconds at 62°C, 20 cycles of 1 second at 95°C and 20 seconds at 62°C, and 14 cycles of 1 second at 95°C and 35 seconds at 62°C. In some embodiments, the denaturation step is omitted for the first cycle after the initial denaturation step. In some embodiments, Taq polymerase is used for amplification. In some embodiments, the cycles are performed at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, or at least 45 times. In some implementations, Taq with hot-start capability is used. In some implementations, the detection of the target nucleic acid occurs within less than 3 hours, less than 2.5 hours, less than 2 hours, less than 1 hour, or less than 30 minutes from initial denaturation to the final extension. In some implementations, the target nucleic acid is detected by methods including real-time quantitative PCR, for example, using FRET probes, such as those described above.

[0272] In some implementations, quantification of real-time PCR results is accomplished by constructing a standard curve from known concentrations of nucleic acids and then extrapolating the quantification information of unknown concentrations of target nucleic acids. In some implementations, the nucleic acid used to generate the standard curve is DNA (e.g., an endogenous or exogenous control). In some implementations, the nucleic acid used to generate the standard curve is purified double-stranded plasmid DNA or in vitro generated single-stranded DNA.

[0273] In some embodiments, for an assay to indicate the absence of a given target nucleic acid in a sample, the Ct values ​​of endogenous controls (such as SAC) and / or exogenous controls (such as SPC) must be within a previously determined valid range. For example, in some embodiments, the absence of a specific target nucleic acid cannot be confirmed unless a control is detected, indicating that the assay is successful.

[0274] In some implementations, a threshold Ct (or “critical Ct”) value for the target nucleic acid (including endogenous and / or exogenous controls) has been previously determined; a value below this value is considered to indicate that the gene has been detected. In some implementations, the threshold Ct is used in conjunction with the assay conditions and system (such as...) employed when the sample is tested. The same measurement conditions and systems (such as) To determine.

[0275] Real-time PCR is performed using any PCR instrument available in the art. Typically, instruments used for real-time PCR data collection and analysis include a thermal cycler, optics for fluorescence excitation and emission collection, and optionally a computer and data acquisition and analysis software.

[0276] In some implementations, the number of target nucleic acids measured exceeds the number of detectable markers, for example, in certain instruments. Therefore, melting analysis can be performed after PCR amplification to increase the number of results that can be reported. Typically, target organisms requiring high sensitivity, such as viruses, can be detected using TaqMan probes or molecular beacon probes via real-time PCR, while target organisms requiring lower sensitivity, such as bacteria, can be detected using melting analysis. However, some viruses, such as metapneumovirus and influenza H1N1, can be detected using melting analysis.

[0277] Another method for detecting target nucleic acids may include high-resolution melting only. For example, The system performs nested multiplex PCR using the following steps: First, reverse transcription is performed, followed by multiple first-stage PCR reactions (PCR1). Then, multiple second-stage PCR reactions (PCR2) are performed simultaneously in the array to amplify the sequences within the PCR1 products. Target nucleic acids can then be detected and analyzed using endpoint melting curve data to generate results for each analyte.

[0278] Target nucleic acids can also be detected via real-time PCR, but in more than one reaction chamber. The QIAstat-Dx analyzer utilizes eight real-time PCR reaction chambers, providing six-fold detection capability. Another method for detecting target nucleic acids can include digital microfluidics or electrowetting and electrochemical detection. For example, the Dx ePlex instrument utilizes digital microfluidics or electrowetting to cause movement and transfer of samples and reagents within the cartridge. The ePlex system includes a microarray for detection, consisting of target-specific capture probes attached to gold electrodes (solid support), which generate a voltage signal if the "target DNA / signal probe" hybridizes with the capture probe. Target nucleic acids can also be detected using chips containing integrated sensor arrays.

[0279] Examples of other methods that can be used with the methods described herein include bead-based flow cytometry assays. See Lu J. et al. (2005) Nature 435:834-838, whose description therein is incorporated herein by reference. An example of a bead-based flow cytometry assay is Luminex, Inc. Technology. See www.luminexcorp.com / technology / index.html. Another approach uses microfluidic devices and single-molecule detection. See U.S. Patents 7,402,422 and 7,351,538 to Fuchs et al., US Genomics, Inc., each of which is incorporated herein by reference in its entirety. Another approach is simple gel electrophoresis and detection with labeled probes (e.g., probes labeled with radiolabels or chemiluminescent labels), such as by Northern blotting.

[0280] In some implementations, methods for detecting target nucleic acids do not include bead-based flow cytometry assays, microfluidic devices and single-molecule detection, simple gel electrophoresis, use of capture probes attached to solid supports, separation of reaction mixtures into multiple reaction chambers, array-based detection, nested amplification, electrochemical detection, high-resolution melting only, or combinations thereof.

[0281] Automated Assay Methods

[0282] Easily automated methods have attracted considerable attention. The methods described herein can be performed in a largely automated manner using commercially available nucleic acid amplification systems. Exemplary non-limiting nucleic acid amplification systems that can be used to perform the methods of the present invention include... system, Infinity system and The Xpress system (Cepheid, Sunnyvale, Calif.). In some implementations, the amplification system can be obtained at the same location as the individual being tested, such as a healthcare provider's office, clinic, or community hospital, thus avoiding delays in processing due to transporting samples to another institution. Assays according to the methods described herein can be performed using automated systems, such as... The system completes its task within 3 hours, within 2 hours in some implementations, within 1 hour in some implementations, within 45 minutes in some implementations, within 35 minutes in some implementations, and within 30 minutes in some implementations. Utilizing a self-contained, disposable kit, sample extraction, amplification, and detection can all be performed within this self-contained sample kit as described herein.

[0283] In some embodiments, after the sample is added to the cartridge, the sample is contacted with a lysis buffer, and the released nucleic acids (NAs) bind to an NA-binding substrate, such as silica or a glass substrate. The sample supernatant is then removed, and the NAs are eluted in an elution buffer, such as Tris / EDTA buffer. The elution buffer can then be processed within the cartridge to detect target nucleic acids as described herein. In some embodiments, the elution buffer is used to reconstitute at least some PCR reagents, which are present in the cartridge in lyophilized granular form.

[0284] A multi-chamber cartridge may contain the primer set and optional probes, or subsets thereof, described herein disposed within the chambers. In some embodiments, the primer set and optional probes, or subsets thereof, described herein are disposed in more than one of the multiple chambers.

[0285] In some embodiments, RT-PCR is used to amplify and analyze the presence of target nucleic acids. In some embodiments, reverse transcription is performed using MMLV and / or CAT-A RT enzymes, incubated at 40°C to 50°C for 5 to 20 minutes. In some embodiments, PCR is performed using a Taq polymerase with hot-start capability, such as AptaTaq (Roche). In some embodiments, initial denaturation is performed at 90°C to 100°C for 20 seconds to 5 minutes; cyclic denaturation is performed at 90°C to 100°C for 1 to 10 seconds; cyclic annealing and amplification are performed at 60°C to 75°C for 10 to 40 seconds; and a maximum of 50 cycles are performed.

[0286] In some embodiments, a double denaturation method is used to amplify low copy number target nucleic acids. In some embodiments, the double denaturation method comprises a first denaturation step followed by the addition of primers and / or probes for detecting the target nucleic acid. Then, all or most of the nucleic acid-containing sample (such as DNA elution buffer) is subjected to a second denaturation, and in some cases, a portion of the sample is subsequently aliquoted for cycling and detection of the target nucleic acid. While not wishing to be bound by any particular theory, the double denaturation protocol can increase the chance that low copy number target nucleic acids (or their complementary strands) will be present in the aliquots selected for cycling and detection because the second denaturation effectively doubles the quantity of the target nucleic acid (i.e., it separates the target nucleic acid from its complementary strand into two separate templates) before the aliquots are selected for cycling. In some embodiments, the first denaturation step comprises heating to a temperature of 90°C to 100°C for a total time of 30 seconds to 5 minutes. In some embodiments, the second denaturation step comprises heating to a temperature of 90°C to 100°C for a total time of 5 seconds to 3 minutes. In some embodiments, the first denaturation step and / or the second denaturation step are performed by heating aliquots of the sample separately. In some embodiments, each aliquot may be heated for the durations listed above. As a non-limiting example, the first denaturation step of a NA-containing sample (such as DNA elution buffer) may comprise heating at least one, at least two, at least three, or at least four aliquots of the sample, respectively (sequentially or simultaneously), to a temperature of 90°C to 100°C for 60 seconds each. As a non-limiting example, the second denaturation step of a NA-containing sample (such as DNA elution buffer) containing enzymes, primers, and probes may comprise heating at least one, at least two, at least three, or at least four aliquots of the elution buffer, respectively (sequentially or simultaneously), to a temperature of 90°C to 100°C for 5 seconds each. In some embodiments, the aliquots are the entire NA-containing sample (such as DNA elution buffer). In some embodiments, the aliquots are less than the entire NA-containing sample (such as DNA elution buffer).

[0287] In some implementations, offline centrifugation is used, for example, for samples with low cell content. The sample is centrifuged with or without buffer added, and the supernatant is removed. The precipitate is then resuspended in a smaller volume of supernatant or buffer. The resuspended precipitate is then analyzed as described herein.

[0288] Exemplary Automation and Systems

[0289] Many existing fully integrated nucleic acid amplification and assay systems capable of sample preparation are typically quite complex and expensive. The nucleic acid amplification and assay system presented in this article provides a rapid, simple, convenient, and economical approach to nucleic acid analysis.

[0290] System Overview

[0291] On one hand, the present invention relates to a sample cartridge utilizing a valve body platform for the detection of both encapsulated and free target nucleic acids. In some embodiments, the valve body includes a sample processing area or lysis chamber providing either or both mechanical and chemical lysis. This allows a single cartridge to lyse multiple different types of targets, and thus can be considered a "combination assay cartridge." In some embodiments, the sample cartridge can process and detect bacterial targets requiring mechanical lysis and viral targets suitable for chemical lysis.

[0292] A sample cartridge device can be any device configured to perform one or more process steps related to the preparation and / or analysis of biological fluid samples according to any of the methods described herein. In some embodiments, the sample cartridge device is configured to perform at least sample preparation. The sample cartridge can also be configured to perform additional processes, such as detecting target nucleic acids in a nucleic acid amplification assay (NAAT), for example, a polymerase chain reaction (PCR) assay, by using a reaction vessel connected to the sample cartridge. In some embodiments, the reaction vessel extends from the cartridge body. The preparation of fluid samples typically involves a series of processing steps, which, depending on the specific protocol, may include chemical, electrical, mechanical, thermal, optical, or acoustic processing steps. Such steps can be used to perform various sample preparation functions, such as cell capture, cell lysis, analyte binding, and binding of unwanted substances.

[0293] The sample cartridge suitable for use in this invention includes one or more delivery ports through which prepared fluid samples can be transported to a connected reaction vessel for analysis. FIG. 1Figure A illustrates an exemplary combined assay cartridge 100, suitable for sample preparation and analysis via PCR when received in an instrument module according to some embodiments. The cartridge is connected to a reaction container 116 (also referred to as a “reaction tube” or “PCR tube”), adapted to analyze fluid samples processed within the cartridge 100. In some embodiments, the reaction container extends from the cartridge body. Such a cartridge 100 includes various components, including a main housing 102 having one or more chambers 108 for processing fluid samples, which typically includes sample preparation prior to analysis. In these embodiments, the cartridge may be a fully integrated nucleic acid amplification and testing system combining sample preparation, amplification, and detection. The instrument module facilitates the processing steps required for sample preparation, and the prepared sample is transported via one of a pair of delivery ports to a fluid conduit connected to the reaction container 116 of the cartridge 100 housing. The prepared biofluid sample is then transported to the reaction chamber of the reaction container, where it undergoes nucleic acid amplification. In some embodiments, the amplification is a polymerase chain reaction (PCR). In some implementations, concurrent with the amplification of the biofluid sample, the excitation and optical detection devices of the module are used to detect optical emission indicating the presence or absence of a target nucleic acid analyte of interest, such as bacteria, viruses, pathogens, toxins, or other target analytes. It should be understood that such reaction containers can include a variety of different chambers, conduits, or micropore arrays for detecting target analytes. The sample cartridge may be equipped with means for preparing the biofluid sample prior to transport to the reaction container. Any chemical reagents required for virus or cell lysis, or devices for binding or detecting the analyte of interest (e.g., reagent beads), can be contained in one or more chambers of the sample cartridge and are therefore available for sample preparation.

[0294] Exemplary use of a reaction vessel for analyzing biological fluid samples is described in commonly assigned U.S. Patent No. 6,818,185, filed May 30, 2000, entitled “Cartridge for Conducting a Chemical Reaction,” the entire contents of which are incorporated herein by reference for all purposes. Examples of sample boxes and related modules are shown and described in U.S. Patent No. 6,374,684, filed August 25, 2000, entitled “Fluid Control and Processing System”; U.S. Patent No. 8,048,386, filed February 25, 2002, entitled “Fluid Processing and Control”; U.S. Patent Application No. 63 / 217,672, filed July 1, 2021, entitled “Universal Assay Cartridge and Methods of Use”; U.S. Provisional Application No. 63 / 319,993, filed March 15, 2022, entitled “Unitary Cartridge Body and Associated Components and Methods of Manufacture”; and U.S. Patent No. 10,562,030, filed July 22, 2016, entitled “Molecular Diagnostic Assay System”; the entire contents of which are incorporated herein by reference for all purposes. The aforementioned patents are included in the appendix.

[0295] Various aspects of the sample cartridge 100 can be further understood by referring to U.S. Patent No. 6,374,684 (“'684 Patent”), which describes certain aspects of the sample cartridge in more detail. Such a sample cartridge may include a fluid control mechanism, such as a rotary fluid control valve assembly, which is fluidly connected to a chamber of the sample cartridge. The term “chamber” is used interchangeably with the terms “orifice,” “tube,” etc. Rotation of the rotary fluid control valve allows fluid communication between the chamber and the valve, thereby controlling the flow of a biological fluid sample deposited in the cartridge into different chambers in which various reagents can be dispensed as needed, according to a specific protocol, to prepare a biological fluid sample for analysis. To operate the rotary valve, the cartridge processing module includes a motor, such as a stepper motor, typically connected to a drive system that engages with a feature of the valve in the sample cartridge to coordinate with the movement of a syringe to control the movement of the valve, thereby causing the fluid sample to move according to a desired sample preparation protocol. The '684 Patent describes the fluid metering and dispensing function of the rotary valve according to a specific sample preparation protocol.

[0296] Exemplary Assay Cartridge and Valve Assembly

[0297] SUMMARY

[0298] like FIG. 1 As shown in Figure A, the combined assay kit 100 includes a housing 102 containing multiple chambers 108 for reagent or buffer solutions and sample processing. The chambers are arranged around a central syringe barrel 106, which is connected to a valve body 110 (see Figure A). FIG. 1 B and FIG. 1 C) Fluid communication is maintained and sealed with gasket 104. Valve body 110 may include cap 112, and the entire housing may be supported on housing base 101. Valve body typically contains one or more channels or cavities (chambers 114), which may contain filters as described herein, serving to bind and elute nucleic acids. In some embodiments, the housing also includes one or more temperature-controlled channels or chambers, which in some embodiments may serve as thermal cycling chambers. A plunger (not shown) can be operated to draw fluid into syringe barrel 106, and rotation of valve body 110 provides selective fluid communication between the various reagent chambers 108 and channels, reaction chambers(multiple), mixing chambers, and optionally any temperature-controlled areas. Thus, the various reagent chambers 108, reaction chambers, filter materials(multiple), and temperature-controlled chambers or channels are selectively fluidly communicated by rotation of the plunger, and reagent movement (e.g., chamber loading or unloading) is operated by the plunger's "syringe" action within the valve assembly. In other embodiments, the individual reagent chambers, reaction chambers, filter materials, and temperature control chambers or channels are selectively fluidly connected by the linear movement of reagents and samples from one chamber to the next (e.g., by forced movement).

[0299] Exemplary Valve Assembly

[0300] FIG. 2 The diagram illustrates what can be used FIG. 1Different valve assemblies for sample cartridge A. Each valve assembly shown is inverted to better illustrate the components of the valve body. Valve assembly A performs only mechanical lysis and is suitable for lysing stubborn targets (e.g., certain bacteria, spores). Valve assemblies B and C perform only chemical lysis and are suitable for lysing less stubborn targets (e.g., viruses, free NA, some spores, some bacteria, and yeast). Valve assembly D can perform both mechanical and chemical lysis on all types of targets. In all such cartridges, the valve assembly includes a syringe tube 106, a valve body 110, and a valve cap 112. On the one hand, the additional capabilities of the valve assemblies of the sample cartridge depend in part on the features of the filter, valve body, and cap, as well as the specific workflow sequence performed by the instrument interface of the module. For example, valve assembly A has a valve body 110 shaped with a circular cavity to support a polymer filter disc 116 for filtering the sample, and a cap 112 with an ultrasonic dome feature 113 that connects to the ultrasonic amplitude transformer interface of the cartridge receiving module for ultrasonic lysis of the target. Conversely, valve assemblies B and C have a valve body 110 with an elliptical filter recess 111 in which a glass filter 114 (e.g., a glass column) is received, the glass filter being configured to bind to nucleic acids released from the target via chemical lysis. Valve assembly D is more similar in design to valve assembly A, having a cap 112 with an ultrasonic dome 113 and a valve body with a circular cavity for supporting a disc filter; however, this design uses a glass fiber filter 117. The filter is formed using glass fibers, which facilitates affinity binding to free nucleic acids released via chemical lysis. Therefore, this filter is suitable for both mechanical and chemical lysis. U.S. Provisional Application No. 63 / 217,672 further details the assay cartridge of valve assembly D, which is incorporated herein by reference in its entirety.

[0301] Reaction Module

[0302] In some embodiments, cartridge 200 is configured for insertion into reaction module 300. The module is configured to receive cartridge 200. In some embodiments, the reaction module includes a heating plate 308 to heat a temperature-controlled chamber or channel. The module may optionally additionally include a fan 304 to provide cooling, where the temperature-controlled channel or chamber is a thermal circulation channel or chamber. Electronic circuitry 302 may be provided to transmit information (e.g., optical information) to a computer for analysis. In some embodiments, the module may include an optical block 306 to provide excitation and / or detection of one or more (e.g., 1, 2, 3, 4, or more) light signals representing, for example, signal DNA amplified against various PCR targets. In various embodiments, an electrical connector 312 may be provided for interfaced with a system (e.g., a system controller) or with discrete analysis / controller units. A pipette 310 can be used to introduce a sample into the cartridge. In some embodiments, the module also includes a controller that operates the rotation of a plunger and valve body in a syringe barrel.

[0303] Analysis System

[0304] In some embodiments, a system (e.g., a processing unit) is provided. The system includes a housing configured to support and power a plurality of sample processing modules 300, each configured to hold and operate a removable cartridge 100. In some embodiments, the system is configured to operate the sample processing modules to perform PCR assays on one or more target nucleic acid analytes and optionally determine the levels of one or more target RNA / DNA sequences within a corresponding removable sample cartridge. Typically, processing of a sample within a corresponding removable sample cartridge involves operating the cartridge to perform the methods described herein. In some embodiments, the system is configured to contain one sample processing module. In some 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). In some embodiments, the system has a user interface that allows a user to input operating instructions and / or monitor the operation of the cartridges to determine the presence and / or quantity of one or more nucleic acids.

[0305] Although the methods described in this article are primarily based on Cepheid Inc. (Sunnyvale, Calif.) The box is described (see example). FIG. 1A), but it should be recognized that, in light of the teachings provided herein, the methods can be implemented on other cartridge / microfluidic systems, including alternative cartridge designs with valve assemblies involving multiple interface connection components, and cartridge bodies with multiple chambers defined by multiple interface connection components to form a cartridge, such as those described in Korean Applications No. 102293717B1 and No. KR102362853B1; cartridges that utilize ultrasound to lyse cells in biological samples, such as those described in International Application No. WO2021 / 245390A1; and cartridges and systems that utilize electrowetting grids for microdroplet manipulation and are configured to detect analytes of interest, such as No. WO2016 / 077341. Those described in International Application A2; cassettes that facilitate the movement of nucleic acids from one chamber to the next by opening an exhaust bladder, such as those described in International Application No. WO2012 / 145730A2; multiplex assay systems comprising multiple thermal cycling units such that each chamber can be heated, cooled, and / or compressed to mix fluids within the chamber or to propel fluids from one chamber to another, such as those described in International Application No. WO2015 / 138343A1; and systems for rapid nucleic acid amplification that facilitate it by aligning flexible portions of the sample cassette to complete temperature cycling for nucleic acid amplification, such as those described in International Application No. WO2017 / 147085A1. Such cassette / microfluidic systems may include, for example, microfluidic systems implemented using soft lithography, micro / nanofibrillated microfluidic systems implemented using hard lithography, etc.

[0306] In one exemplary embodiment, the cartridge may include multiple housings, such as a first housing, a second housing, a central syringe barrel in fluid communication with the first and second housings, a reaction vessel, etc. The first housing may be formed of multiple chambers separate from each other for reagent or buffer and sample processing. In some embodiments, the first housing may be used for storing multiple reagents. The second housing may be formed of one or more chambers separate from each other and includes a path through which reagents or samples from the first housing move. When the first and second housings of the cartridge are combined, a liquid flow path and optionally an air flow path may be formed between the two chambers via the central syringe barrel. The liquid flow path is connected to the first housing to provide space for the movement and mixing of samples and reagents. The air flow path may connect the reaction vessel and a vacuum control area of ​​a “plunger” to control any vacuum that may occur when extracted nucleic acids move into the reaction vessel. Rotation of the syringe barrel containing the “plunger” can sequentially draw samples and reagents from the multiple chambers into the internal space of the syringe barrel and expel the mixture from the internal space into any of the multiple chambers of the cartridge (the first or second housing). The rotation of the syringe barrel containing the "plunger" draws reagents from the multiple chambers of the cartridge into the internal space of the syringe barrel, and then discharges the mixed reagents into the nucleic acid amplification reaction container.

[0307] In another exemplary embodiment, the cartridge includes a flow channel cap and a substrate, which together form a closed channel therein. In one embodiment of this configuration, an inner chamber containing the reagents required for dielectric extraction is separated from the outer chamber, and the upper and lower portions of the inner chamber are sealed. Additionally, a double-layered flow channel cap-pad structure can be provided between the outer chamber and the substrate. A closed flow path is formed by achieving a strong connection between the substrate-flow channel cap-pad-outer chamber. In this configuration, microbeads required for dielectric extraction and amplification are also provided, which are housed in a double-chamber structure of outer chamber-microbead chamber. Even when the microbead chamber is open, the microbeads can be maintained by a dehumidification unit located above the microbead chamber.

[0308] In a further exemplary embodiment, the cartridge may include multiple reaction chambers; more specifically, the reaction container may include multiple reaction chambers. In these embodiments, different types of lyophilized primers and probes may be provided in each reaction chamber. For example, primers and probes for virus-associated nucleic acids may be provided in one reaction chamber, and primers and probes for bacterial-associated nucleic acids may be provided in a second reaction chamber for amplification and detection, and so on. Of course, various amplification and detection processes can be performed simultaneously in a single reaction chamber. Therefore, the amplification of each target nucleic acid described herein may be performed individually in separate reaction chambers or wells, or in a single reaction chamber or well as a multiplex reaction.

[0309] Furthermore, it should be understood that the combined assay methods described herein (i.e., identifying multiple diseases based on comparative levels of multiple target nucleic acids obtained from a single sample) can be further implemented in entirely different systems, including: isothermal nucleic acid amplification systems, digital RT-PCR, electrochemical PCR, lateral flow assay kits, electrochemical sensors, nucleic acid sequencing, CRISPR / Cas-based technologies, chemiluminescence, and nanoparticle-based colorimetric detection.

[0310] In various implementations, signal DNA from a PCR (nucleic acid amplification) reaction is amplified for detection and / or quantification. In some implementations, amplification includes any of a variety of methods, including but not limited to polymerase chain reaction (PCR), ligase chain reaction (LCR), ligase detection reaction (LDR), multiplex ligation-dependent probe amplification (MLPA), post-ligation Q-replicaase amplification, primer extension, strand displacement amplification (SDA), superbranched strand displacement amplification, multiple displacement amplification (MDA), nucleic acid chain-based amplification (NASBA), rolling circle amplification (RCA), etc.

[0311] In illustrative but non-limiting embodiments, the amplification reaction may generate a light signal proportional to the amount of target nucleic acid (e.g., signal DNA) amplified. Illustrative light signals include, but are not limited to, fluorescence signals, chemiluminescence signals, electrochemiluminescence signals, colorimetric signals, etc. In some embodiments, the light signal is a fluorescent light signal generated by a fluorescent indicator. In some embodiments, the fluorescent indicator is a non-specific intercalating dye that binds to the double-stranded DNA product, while in some other embodiments, the fluorescent indicator comprises a target sequence-specific probe (e.g., probe, probe, MOLECULAR wait).

[0312] A single PCR reaction (nucleic acid amplification) or multiple PCR reactions (nucleic acid amplification) can be run sequentially (or simultaneously in separate temperature-controlled channels or chambers) using the same detectable marker, because sequentially run PCR signal DNA is analyzed sequentially, and simultaneously run PCR signal DNA is distinguished by its appearance in different temperature-controlled channels or chambers. The signal produced by this amplification can be distinguished from other amplification products because it was not run simultaneously and / or because it ran in different reaction channels / chambers. However, in the case of multiple nucleic acid amplifications running simultaneously in the same chamber, it is generally common practice to detect and / or quantify each analyzed reaction product by using different and distinguishable markers.

[0313] In some embodiments, the amplified products (amplified nucleic acids from a nucleic acid assay) can be detected using methods well known to those skilled in the art. In some embodiments, amplification is a direct and simple PCR amplification reaction. However, in some embodiments, nested PCR reactions are used to amplify nucleic acids from a nucleic acid assay. Multiplex PCR assays are considered in various embodiments, particularly where it is necessary to analyze multiple products of a nucleic acid assay in the same amplification reaction. In some embodiments, in such multiplex amplification reactions, each probe (e.g., for each specific analyte) has its own specific dye / fluorophore, making it detectable independently of other probes. In some embodiments, typically, to generate a signal, probes used in various amplification reactions utilize fluorescence changes in a fluorophore for detection and / or quantification of the amplified products, said changes being due to alterations in the interaction between the fluorophore and another molecule or moiety, the alteration of which is caused by changes in the distance between the fluorophore and the interacting molecule or moiety. Alternatively, other methods for detecting polynucleotides in a sample are considered, including but not limited to the use of radiolabeled probes.

[0314] Assay Workflow

[0315] Before amplification, the sample undergoes one or more sample preparation operations. Typically, these operations include manipulations such as: extracting intracellular material, e.g., nucleic acids, from whole-cell samples, viruses, etc., to form a crude extract; and additional treatments to prepare the sample for subsequent operations, such as denaturation, purification, filtration, desalting, etc., of contaminating (e.g., DNA-binding) proteins. Release of nucleic acids from sample cells or viruses, and denaturation of DNA-binding proteins, can typically be performed by chemical, physical, or electrolytic lysis methods. For example, chemical methods typically employ lysis reagents to disrupt cells and extract nucleic acids, followed by treatment of the extract with plasma salts such as guanidine isothiocyanate or urea to denature any contaminating and potentially interfering proteins. Typically, when using chemical extraction and / or denaturation methods, appropriate reagents can be added to the sample preparation chamber, a separate accessible chamber, or introduced externally. Preferably, sample preparation is performed in only one step or no more than two steps. For example, sample preparation may include heating the sample in a lysis solution without further purification prior to amplification. In some embodiments, the lysed sample may be diluted prior to amplification. One or more of these different sample preparation procedures can be easily incorporated into the fluid closed-box system considered in this paper.

[0316] FIG. 5A -C illustrates an illustrative but non-limiting workflow for nucleic acid amplification. On one hand, the assay kits described herein are capable of performing specialized workflows that lyse and detect different target analytes required for specific combination assays. In some embodiments, the kit is configured for chemical lysis of multiple target organisms. In other embodiments, the kit is configured for mechanical lysis of multiple target organisms. In still other embodiments, the kit is configured for both mechanical and chemical lysis to achieve lysis of multiple different types of targets. Therefore, the kit can be configured for combination assays via existing workflows associated with conventional kits, or it can be operated according to new workflows specifically configured for combination assays.

[0317] On the one hand, it has the characteristics of this article FIG. 2 The sample cartridge of the valve assembly described in D is capable of performing various workflows, which involve: chemical lysis of the target organism, mechanical lysis of the target organism, or both. Therefore, the sample cartridge can perform existing workflows associated with conventional dedicated cartridges, or it can perform entirely new workflows that involve both types of lysis.

[0318] Figure 5 illustrates exemplary assay workflows that can be performed using a single universal cartridge according to some embodiments. In any of these embodiments, the filter may be formed of a glass filter to facilitate affinity binding of nucleic acids (NAs) to the glass fibers, and the pore size may also be suitable for chemical lysis. In any of these workflows, nucleic acid amplification may be PCR, real-time PCR, isothermal amplification (including but not limited to nucleic acid sequence-based amplification, loop-mediated isothermal amplification, helicase-dependent amplification, rolling circle amplification, multiple displacement amplification, whole genome amplification, or recombinase polymerase amplification), or other nucleic acid amplification methods known to those skilled in the art.

[0319] In workflow A, the sample is optionally exposed to sample processing or chemical lysis, and then the processed or lysed fluid sample is passed through a filter in which target organisms are captured. In some embodiments, sample processing is used to weaken cell walls or inactivate the sample or make it less viscous to facilitate processing through the filter. The filter is then washed, leaving the target organisms on it. Next, the target organisms are mechanically lysed, such as by sonication, to release nucleic acids (NA). In some embodiments, mechanical lysis includes packing glass microbeads along the inside of the filter to aid in the mechanical lysis of the target. The filter then elutes the NA, followed by nucleic acid amplification.

[0320] In workflow B, the sample is chemically lysed to obtain the NA target. In some embodiments, after chemical lysis, the NA binds to a filter via precipitation and the presence of a binding reagent. The filter is then washed with a rinsing reagent while retaining the NA bound to it. Typically, the washing reagent contains a certain amount of salt, which still promotes NA binding to the filter while allowing the removal of non-target substances. The filter is then eluted to remove the NA target. In some embodiments, elution is performed with a pH-neutral buffer or an alkaline buffer. The target NA is then delivered to an attached reaction vessel for nucleic acid amplification.

[0321] In workflow C, the fluid sample is exposed to sample processing and / or chemical lysis of the target organism. The NA released by chemical lysis then binds to a filter. This step may utilize precipitation and binding reagents. The filter is then washed with a rinsing reagent while maintaining the NA binding. Typically, the washing reagent contains a certain amount of salt, which still promotes NA binding to the filter while allowing the removal of non-target material. Next, the target organism trapped in the filter is thermally and / or mechanically lysed. This may utilize sonication, and glass microbeads may also be used to facilitate mechanical lysis of the selected target organism. The lysed target NA is then eluted from the filter. In some embodiments, elution is performed with a pH-neutral buffer or an alkaline buffer. The target NA is then delivered to an attached reaction vessel for nucleic acid amplification. Therefore, in this workflow, the lysis of various target organisms is achieved, some requiring only chemical lysis (e.g., viral targets), while others require mechanical lysis (e.g., bacteria, spores, etc.), so that all these target NAs can be released from a single sample and tested by the same sample cassette. Although the above workflow describes chemical lysis followed by mechanical lysis, it should be understood that other workflows in which mechanical lysis is performed first followed by chemical lysis can also be used.

[0322] In some implementations, the sample cartridge includes an identifier carrying appropriate workflow information about the specific combination to be measured, causing the instrument module receiving the sample cartridge to operate according to the specified workflow.

[0323] Exemplary Assay Configuration

[0324] Reagents for Respiratory Biomarker Panel Assay

[0325] FIG. 6 Showing what can be used as An exemplary sample cartridge, as part of the system, has various compartments numbered. The exemplary cartridge may include: lyophilized reagents in the form of one or more microbeads as described herein; optional lysis reagents; an alkali; optional binding reagents; a filtration reagent; a washing reagent; and an elution reagent. The last four reagents are named according to the function they perform on nucleic acids. Thus, for example, binding reagents facilitate the binding of nucleic acids to substrates, and filtration reagents facilitate the filtration of nucleic acids.

[0326] In some embodiments, the lysis reagent may include a dissociating agent, a chelating agent, a buffer, an alkaline agent, or a detergent. The dissociating agent may be selected from guanidine compounds such as guanidine thiocyanate or guanidine hydrochloride, alkali metal perchlorates such as lithium perchlorate, alkali metal iodides, magnesium chloride, urea, thiourea, formamide, or combinations thereof. The concentration of the dissociating agent may be in the range of about 1 M to about 10 M, such as about 2.5 M to about 7.5 M, or less than 4.5 M, less than 2 M, or less than 1 M. The chelating agent may be selected from N-acetyl-L-cysteine, ethylenediaminetetraacetic acid (EDTA), diethylenetriaminepentaacetic acid (DTPA), ethylenediamine-N,N'-disuccinic acid (EDDS), 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA), and phosphonate chelating agents. The concentration of the chelating agent may be in the range of about 10 mM to about 100 mM, and / or about 0.5% to about 5% of the lysis reagent. The buffer solution can be selected from Tris, phosphate buffer, PBS, citrate buffer, TAPS, Bicine, Tricine, TAPSO, HEPES, TES, MOPS, PIPES, cacodylate, SSC, and MES. The concentration of the buffer solution can range from about 5 mM to about 100 mM, such as from about 5 mM to about 50 mM. The detergent can be selected from ionic or nonionic detergents. In some examples, the detergent contains detergents selected from: N-lauroyl sarcosine, sodium dodecyl sulfate (SDS), and hexadecyl methyl ammonium bromide (CTAB). -X-100, n-octyl-β-D-glucopyranoside, CHAPS, n-octyl sucrose, n-octyl-β-D-maltopyranoside, n-octyl-β-D-thiopyranoside F-127, 20, and n-heptyl-β-D-glucopyranoside. The detergent may comprise about 0.1% to about 2% of the lysis reagent, and / or in the range of about 10 mM to a maximum of about 100 mM. The pH of the lysis reagent may be in the range of about pH 3.0 to about pH 5.5.

[0327] In some embodiments, the assays disclosed herein do not utilize a clinker or lysis buffer. When a clinker or lysis buffer is not used, the sample may be contacted with a buffer (or filter reagent) comprising, for example, saline (including one or more inorganic salts such as CaCl2, MgSO4, KCl, NaHCO3, NaCl, etc.), phosphate buffer, Tris buffer, 2-amino-2-hydroxymethyl-1,3-propanediol, HEPES, PBS, citrate buffer, TES, MOPS, PIPES, dimethylarsyl salt, SSC, MES, sugars or disaccharides, or combinations thereof. For example, the buffer may be a commercially available buffer, such as Hanks balanced salt solution available from Sigma Aldrich or TE buffer available from Fisher BioReagents.

[0328] In some embodiments, the alkali agent may be selected from alkali metal hydroxides, such as sodium hydroxide or potassium hydroxide. The concentration of the alkali agent may be from about 0.5N to 5N.

[0329] Binding agents can facilitate the binding of nucleic acids to filters, thereby promoting the removal of non-target substances. In some embodiments, binding agents may include binding polymers such as polyacrylic acid (PAA), polyacrylamide (PAM), polyethylene glycol (PEG), poly(sulfobetaine), or salts, or combinations thereof. In some embodiments, filtering and / or washing agents may include binding agents. For example, binding agents, filtering and / or washing agents may include binding polymers (e.g., PEG200), buffers, inorganic salts, antioxidants and / or chelating agents, defoamer SE15, sodium azide, disaccharides or disaccharide derivatives, carrier proteins, detergents, or DMSO. The conjugated polymer may be present in amounts of at least 10% v / v, at least 20% v / v, at least 30% v / v, and / or less than 60% v / v, less than 40% v / v, less than 30% v / v, less than 20% v / v, or less than 10% v / v of the conjugating agent, filtration agent, and / or washing agent, or may fall within any range defined by any of these values, such as 10% to 60% v / v. The buffer may be selected from Tris, 2-amino-2-hydroxymethyl-1,3-propanediol, HEPES, phosphate buffer, PBS, citrate buffer, TAPS, Bicine, Tricine, TAPSO, HEPES, TES, MOPS, PIPES, dimethylarsyl salt, SSC, and MES. The concentration of the buffer may be in the range of about 5 mM to about 100 mM, such as about 5 mM to about 50 mM. Salts such as NaCl, KCl, or MgCl2 may be present at concentrations 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 a reagent selected from: N-acetyl-L-cysteine, ethylenediaminetetraacetic acid (EDTA), diethylenetriaminepentaacetic acid (DTPA), ethylenediamine-N,N'-disuccinic acid (EDDS), 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA), and phosphonate chelating agents. In some embodiments, the antioxidant and / or chelating agent comprises EDTA. In some embodiments, the antioxidant and / or chelating agent comprises from 0.2% to about 5%, from about 0.2% to about 3%, or from about 0.5% to about 2%, or about 0.5% of the binding agent, filtration agent, and / or washing agent. In some embodiments, the concentration of antioxidants and / or chelating agents in the binding agent, filtering agent, or washing agent is in the range of about 2 mM to about 50 mM or about 5 mM to about 20 mM. In some embodiments, the detergent is an ionic detergent or a nonionic detergent. The detergent may be selected from ionic detergents or nonionic detergents. In some examples, the detergent contains detergents selected from: N-lauroyl sarcosine, sodium dodecyl sulfate (SDS), hexadecyl methyl ammonium bromide (CTAB). -X-100, n-octyl-β-D-glucopyranoside, CHAPS, n-octyl sucrose, n-octyl-β-D-maltopyranoside, n-octyl-β-D-thiopyranoside F-127, 20. Brij-35, and n-heptyl-β-D-glucopyranoside. The detergent may comprise about 0.1% to about 2% of the binding agent, filtration agent, and / or washing agent, and / or in the range of about 10 mM to about 100 mM. The pH of the binding agent, filtration agent, and / or washing agent may be in the range of about pH 6.0 to about pH 8.0 (such as about 6.5 to about 7.5).

[0330] In some implementations, the pH of the elution reagent can be greater than about 9, greater than about 10, greater than about 11, or greater than about 12. Using high pH to elute nucleic acids such as DNA is particularly unique to the kit described herein and provides improved speed and performance for the disclosed method. Speed ​​is provided by the rapid neutralization of acidic ammonium ions by a high concentration of hydroxide ions. Alkylamines have a pKa of ~10⁻¹¹ and are immediately deprotonated at pH 12.7 to form a neutral free base on the solid surface and release cationic DNA. Another advantage of high pH is the denaturing effect of KOH on the captured DNA or RNA. Acidic functional groups in the heterocyclic bases of DNA or RNA are immediately deprotonated and cannot form Watson-Crick bonds. Double-stranded structures and other secondary structures are disrupted but can be renatured upon neutralization, for example, with Tris HCl. This chemical denaturation of captured genomic DNA can be advantageous for isothermal assays that do not undergo the routine thermal denaturation required for PCR. The kits provided herein enable the rapid neutralization of eluted DNA or RNA in KOH, followed by reaction with Tris to produce a final pH of approximately 8.5 for downstream PCR or other nucleic acid assays. In some embodiments, the pH of the elution reagent may be less than about 9, less than about 8.5, or less than about 8. This lower pH elution of bound DNA or RNA can be advantageous, especially for devices where rapid neutralization with KOH solution is not readily available. RNA is known to be hydrolyzed at high pH, ​​but short-term exposure to KOH can provide RNA of good quality. In some instances, the elution reagent comprises polyanionic, polycationic, ammonia, or alkali metal hydroxides. For example, the elution reagent may contain polyanionic compounds such as carrageenan, carrier nucleic acids, or combinations thereof.

[0331] In some cases, to reduce bubble formation in one or more chambers, the detergent Brij may be added to one or more of the reagents described herein.

[0332] It should be understood that various other reagents and initial volumes can be used for automated PCR combinatorial assays of samples in the insert cassette.

[0333] Specialized Cartridge Components

[0334] FIG. 6 An exemplary cartridge suitable for performing multi-target combination assays as described herein is shown. The cartridge shown is based on... The box (Cepheid, Inc., Sunnyvale, Calif.). Box 100 includes a box body 102 having a plurality of chambers 108 defined therein for containing various reagents and / or buffer solutions. The chambers are arranged around a central syringe barrel 109, which is in fluid communication with a valve body 110 via a valve syringe tube 106 extending through the syringe barrel 109. In some embodiments, the valve assembly includes any of the valve assemblies described herein, or any suitable valve assembly. In this embodiment relating to the box, the box utilizes a chemically cleavable valve assembly, such as… FIG. 2 Any of the valve assembly BD. Valve body 110 is interfaced into the housing and supported on housing base 101. The housing typically contains one or more channels or cavities, which may contain filter materials as described herein (e.g., glass filter columns) that can bind and elute nucleic acids. In various embodiments, the housing also includes one or more temperature-controlled channels or chambers, which in some embodiments may serve as thermal cycling chambers. A plunger (not shown) can be operated to draw fluid into syringe barrel 109, and rotation of the valve body / syringe tubing provides selective fluid communication between the various reagent chambers and channels, and between the (multiple) reaction chambers. Thus, the various reagent chambers, reaction chambers, (multiple) matrix materials, and channels are selectively fluidly connected by rotation of the valve and plunger, and reagent movement (e.g., chamber loading or unloading) is operated by the "syringe" action of the plunger. The connected reaction vessel 116 is provided with an optical window to provide real-time detection of, for example, amplification products, base identity in sequencing operations, through the operation of modules within the system described herein.

[0335] Although the above method is for The specific compartments within the kit are described, but it should be recognized that specific reagent / compartment dispensing can vary depending on the characteristics of the nucleic acid detection / quantitative assay. It will also be appreciated that, in some implementations, additional considerations are also taken into account. Variations of the cartridge. Such variations may include, but are not limited to, more or fewer reagent chambers and / or chambers of different sizes, two (or more) sample receiving chambers, two (or more) temperature-controlled channels or chambers, stackable cartridges (providing control of both cartridges via a single module), etc. On one hand, the sample cartridge includes one or more features or components specifically configured to meet the unique requirements of a particular multi-target assay. In this embodiment, the sample cartridge utilizes certain components specifically developed for respiratory combination assays.

[0336] FIG. 8 A-8B shows a detailed view of a valve assembly in a multi-target combination assay kit according to some embodiments. In this embodiment, the valve assembly is configured for chemical lysis. The valve assembly includes a valve body 110 having a disc shape and a tubular syringe-type 106 along its proximal portion. The disc portion has a generally flat external upper port surface 120 having one or more ports (e.g., 142, 146), such as... FIG. 7 As shown in Figure A, the valve assembly is rotatable relative to the housing, for example, through engagement with the module's drive mechanism via interface feature 123. Ports 142 and 146 on the top flat surface allow fluid communication between the syringe tubing 106 and each chamber and / or one or more channels within the housing. In this embodiment, ports 142 and 146 interface with a circular washer 126 having a plurality of ports defined therein, which interface with and are fluidly connected to corresponding ports at the bottom of the chambers within the housing. As the valve assembly rotates, the top ports 142 and 146 interface with different pairs of ports to fluidly connect the individual chambers to each other or to channels connected to the reaction vessel of the housing, such that movement of a "plunger" through the syringe tubing propels fluid between the individual chambers and / or channels to facilitate the handling of fluid samples and subsequent analytical testing.

[0337] like FIG. 7 As seen in Figure B, valve body 110 includes a lysis chamber 121 on its lower side, which may contain a solid material for capturing cells, spores, viruses, or microorganisms to be lysed. Suitable solid materials include, but are not limited to, filters, microbeads, fibers, membranes, filter paper, glass wool, polymers, or gels. One or more internal ports facilitate fluid inflow and outflow from the lysis chamber. In this embodiment, the solid material is a glass filter 114 located in a filter recess 111 within valve body 110 for capturing nucleic acids released by the chemical lysis of the target organism in the fluid sample.

[0338] like FIG. 8 As shown in A-8B, the outer wall 122 of the valve body and the bottom cap 112 together form a pyrolysis chamber 121. FIG. 7As shown in Figure B, the lysis chamber 121 includes a pair of processing ports to facilitate the inflow and outflow of fluid samples from the lysis chamber. A first fluid processing port 124 is connected to a first fluid processing channel defined within a valve body, and a second fluid processing port 125 is connected to a second fluid processing channel defined within a valve body. Through these channels, the first fluid processing channel is connected to both the internal conduit and the external port 142 of the syringe tubing 106, while the second fluid processing channel is connected to a second external port 146. With this configuration, the pressure applied by the movement of the plunger through the syringe tubing 106 acts like a syringe, drawing fluid from one chamber into the syringe tubing, and after the valve assembly is rotated to the position where each port is connected to another chamber, the movement of the plunger propels fluid through the valve body and channels into another chamber or channel leading to the reaction vessel. This method can perform various processing steps required for sample preparation protocols for target analytes in multi-target combination assays.

[0339] On one hand, the respiratory tract assembly described herein performs chemical lysis of target bacteria and viruses. Typically, in conventional sample cassettes and methods, these bacterial targets are mechanically lysed (e.g., by sonication), while chemical lysis is usually reserved for less robust targets, such as viruses. To perform chemical lysis of both bacterial and viral targets, the lysis buffer may have increased alkalinity (e.g., sodium hydroxide). Additional high-alkalinity elution buffers (e.g., ammonia or alkali metal hydroxides) can be used to elute nucleic acids bound to the glass filter. While these buffers enable the chemical preparation of the sample, in practice, using such buffers in conventional cassettes can be problematic because these high-alkalinity buffers can degrade the sealing interface between the valve assembly material and the cassette components, leading to valve assembly cracking and leakage during processing. Such leakage can be detrimental to sample handling. Dedicated valve assemblies can be used to resist the high alkalinity of these buffers (e.g., greater than pH 10, greater than pH 11, or greater than pH 12). One challenge in developing conventional valve assemblies is that these assemblies are typically manufactured by injection molding of certain polymer materials, usually polycarbonate, polyolefins (including polyethylene or polypropylene), or combinations thereof. Such typical polymer materials used in injection molding are not resistant to the aforementioned increased alkalinity range, while resistant polymer materials can be significantly more expensive and may be less suitable for injection molding small-sized microfluidic features. Therefore, specialized valve assemblies can be developed by annealing valve body assemblies formed from conventional polymers to sufficiently harden the polymer material to resist increased alkalinity. Annealing polycarbonate reduces the chemical corrosion of the mixture of NaOH and GTC in pyrolysis and elution reagents. In some embodiments, after valve assembly formation, they are heated to an increased temperature (e.g., 90-100°C, approximately 100°C) for about an hour or longer, and then slowly cooled in a temperature-controlled manner for at least 30 minutes. Studies have shown that these annealed valve assemblies result in valve assemblies with essentially the same design and materials as conventional valve assemblies, except that they are resistant to high-alkalinity buffers that may be impractical in conventional cases. The alkali resistance of the annealed valve body can be tested by exposing the valve body assembly (VBA) to NaOH / GTC and then visually inspecting for cracks in the VBA after approximately 30 minutes. It should be understood that such annealing can be advantageous for various reasons and is not necessarily tied to any specific assay described herein. Furthermore, it should be understood that the assay described herein can be performed with various other box designs, equipment, and systems and is not necessarily tied to the specific box design described herein.

[0340] Exemplary Detection Methods, Results, and Result Processing

[0341] In some embodiments, a computer-based analysis program is used to convert the raw data generated by a detection assay into data that is predictive for a clinician. In an illustrative automated embodiment, the analyte names for pathogens and detection methods are listed in Table C.

[0342] Table C: Respiratory Biomarker Panel Targets

[0343] Targets Analyte Name Detection Method Adenovirus Adenovirus Amplification Coronavirus Coronavirus Amplification SARS-CoV-2 SARS-CoV-2 Amplification Influenza A Flu A Amplification Influenza A H1N1 09 Flu A H1N1-09 Melting Influenza B Flu B Amplification Human Metapneumovirus hMPV Melting Parainfluenza Virus Parainfluenza Amplification Rhinovirus / Enterovirus Rhinovirus-Enterovirus Amplification and Melting Respiratory Syncytial Virus RSV Amplification Bordetella parapertussis Bordetella parapertussis Melting Bordetella pertussis Bordetella pertussis Melting Chlamydia pneumoniae Chlamydia pneumoniae Melting Mycoplasma pneumoniae Mycoplasma pneumoniae Melting

[0344] By software evaluates the melting curve analysis to determine the presence of PCR products. The melting temperature (T M ) and melting peak height of the curve are automatically calculated by the analysis software. If T m falls within the valid T m range specified for each target nucleic acid, the melting curve is detected as positive. If the melting curve is not within the appropriate T <​​​​​​​​​​​​​​​​A positive result typically indicates that the target has a Ct within the effective range and an endpoint above the threshold setting, and / or that the target has a melting peak above the threshold setting and a melting temperature (Tm) window within the effective range; SPC can be ignored when amplification occurs; all probe checks are satisfactory; and no other pathogens listed in Table 1 are detected except for the target indicated as positive.

[0349] Exemplary Detection Methods for One to Forty Targets

[0350] As described herein, respiratory (or other viral, bacterial, fungal, or parasitic) pathogens can be detected by nucleic acid amplification in a multiplex amplification reaction, which can be designed to detect 1 to 40 (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39) or more target nucleic acids in each amplification reaction mixture. This document discloses a method for identifying up to forty target nucleic acids in biological samples collected from subjects. The method for identifying up to forty target nucleic acids in biological samples collected from subjects may include providing a sample kit as described herein. The sample cartridge preferably comprises multiple chambers, including a lysis chamber having at least one fluid outlet in fluid communication with another chamber. The lysis chamber is adapted for mechanical and chemical lysis to release nucleic acids from a biological sample. The sample cartridge also preferably contains lyophilized and liquid reagents in two or more of the multiple chambers. The lyophilized reagents contain at least one primer set for amplifying and detecting multiple target nucleic acids, and the liquid reagents include lysis buffer, binding reagent, and elution buffer. The sample cartridge also preferably includes a reaction vessel fluidly connected to the multiple chambers of the sample cartridge and configured for amplifying the target nucleic acids.

[0351] Methods for identifying up to forty target nucleic acids in biological samples collected from subjects also include contacting the nucleic acids from the biological sample with at least one primer set. The number of primer sets present depends on the number of target nucleic acids being identified. For example, a sample cassette may include 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40 or more primer sets. Each primer set contains a forward primer, a reverse primer, and a probe specific to the target nucleic acid in the biological sample. As described herein, (multiple) primer sets may be present in the sample cassette in the form of lyophilized microbeads. More than one primer set may be present in each microbead. For example, each lyophilized microbead may include 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40 or more primer sets. The probes in each set may contain a detectable portion for detection via real-time PCR or melting curve analysis.

[0352] Within each primer set, at least one of the forward or reverse primers may contain one or more modified bases. Modified bases may be selected from unstable bases, stable bases, or combinations thereof. "Stable bases" include, for example, fragments of peptide nucleic acids (PNAs) that can be incorporated into DNA oligonucleotides to improve duplex stability. Locked nucleic acids (LNAs) and unlocked nucleic acids (UNAs) are analogs of RNA that can be readily incorporated into DNA oligonucleotides during solid-phase oligonucleotide synthesis and, respectively, improve and decrease duplex stability. Suitable stable bases also include modified DNA bases that improve the stability of base pairs (and thus the overall stability of the duplex). These modified bases can be incorporated into oligonucleotides during solid-phase synthesis and provide a more predictable method for improving DNA duplex stability. Examples include AP-dC (G-clamp) and 2-aminoadenine, as well as 5-methylcytosine and C(5)-propynylcytosine (in place of cytosine), and C(5)-propynyluracil (in place of thymine). Stable bases can increase the melting temperature of primers in double-stranded form or reduce secondary structure formation. "Unstable bases" are bases that destabilize double-stranded DNA by forming base pairs that are less stable than typical AT and / or GC base pairs. Unstable bases can be selected from sterically hindered bases that reduce nonspecific primer interactions, bases that inhibit the melting temperature of primers in double-stranded form, bases that inhibit the elongation of unwanted primer-dimers, or combinations thereof. Inosine (I) is an unstable base because it pairs with cytosine (C), but IC bases are less stable than GC base pairs. This lower stability stems from the fact that inosine is a purine that can only form two hydrogen bonds compared to the three hydrogen bonds in GC base pairs. Other unstable bases are known to those skilled in the art or readily identifiable by them.

[0353] In some instances, primers and / or probes include modified bases that reduce unwanted hybridization. Unwanted hybridization between any two oligonucleotides in an assay can be reduced or prevented by including pseudo-complementary bases in the oligonucleotides. Pseudo-complementary bases are described as “modified bases” as used herein. Modified bases useful in primers and other oligonucleotides described herein include those base pairs in which the modified base forms a stable hydrogen bond with the native complementary base but not with its modified complementary base (e.g., a pseudo-complementary base) (for ease of discussion, the complementary base is also referred to herein as a “partner”). This is achieved in some embodiments when the modified base can form two or more hydrogen bonds with its native partner, but only one hydrogen bond or no hydrogen bonds with its modified partner. This enables the generation of primers and other oligonucleotide pairs that do not form substantially stable hydrogen bonds with each other, as exhibited at melting temperatures below about 40°C (under physiological or substantially physiological conditions). However, the primers of the primer pair form substantially stable hybrids with the complementary nucleotide sequence in the template strand (e.g., the first template strand) of the single- or double-stranded target nucleic acid and with the strand complementary to the template strand (e.g., the second template strand). In some embodiments, hybrids formed with the primers of the present invention are more stable than hybrids formed with primers containing unmodified bases because the number of hydrogen bonds in such hybrids is increased (in some embodiments, doubled). Pseudo-complementary bases are described in U.S. Patent Publication No. 2023 / 0167490A1, the description of which is incorporated herein by reference.

[0354] Typically, compared to interprimer annealing, incorporating a sufficient number of modified nucleotides into the primers described herein preferentially increases the annealing of the primer with the target nucleic acid template strand. To achieve this, it is not necessary to replace every native nucleotide of the primer with a modified nucleotide. In some embodiments, the primer includes, in addition to one or more modified nucleotides, one or more naturally occurring nucleotides and / or variants of naturally occurring nucleotides, provided that these variants do not significantly interfere with the complementary binding ability of the primer, as discussed above. For example, primers including modified nucleotides may include a furanylpentose moiety other than ribose or 2-deoxyribose, and derivatives of ribose and 2-deoxyribose, such as 3-amino-2-deoxyribose, 2-fluoro-2-deoxyribose, and 2-OC... 1-6 Alkyl or 2-O-allyl ribose, especially 2-O-methylribose. The glycosidic bond can be α or β configuration. If desired, the phosphate backbone of the primer can include phosphate thioester bonds.

[0355] The appropriate class of modified A analogue A* is shown as a 3'-phosphate ester (or thiophosphate ester) incorporated into the primer, the general structure of which is provided by the following formulas 1, 2 and 3, wherein: X is N or CH; Y is O or S; Z is OH or CH3; R is H, F or OR2, wherein R2 is C 1-6 Alkyl or allyl, or H in the case of RNA; and R1 is C. 1-4 Alkyl, C 1-4 alkoxy, alkylthio, F or NHR3, where R3 is H or C 1-4 Alkyl group. An illustrative embodiment of A* has 2,6-diaminopurine (2-aminoadenine) as a base. Suitable class of modified T analogs T* are shown as 3'-phosphates (or thiophosphates) incorporated into the primer, the general structure of which is provided by Formula 4, wherein: Y, Z, and R are as defined above; and R4 is H, C 1-6 Alkyl, C 1-6 alkenyl or C 1-6 Alkyne group. An illustrative embodiment of T* has 2-thio-4-oxo-5-methylpyrimidine (2-thiothymine) as a base. When applicable, the latter nucleotide may be abbreviated as 2-sT or d2-sT. Suitable classes of modified G-analytes G* are shown as 3'-phosphates (or thiophosphates) incorporated into the primer, the general structures of which are provided by Formulas 9, 10, and 11, wherein: R1 is H, C 1-4 Alkyl, C 1-4 Alkoxy, C 1-4 Alkylthio, F, or NHR3, wherein R3 is as defined above; and X, Y, Z, and R are as defined above. An illustrative embodiment of G* has 6-oxo-purine (hypoxanthine) as a base. Suitable class of modified C analogs C* are shown as 3'-phosphates (or thiophosphates) incorporated into the primer, the general structure of which is provided by Formula 8, wherein: Y, Z, R, and R4 are as defined above; Z1 is O or NH; and R5 is H or C. 1-4An illustrative embodiment of alkyl. C* has pyrrolo[2,3-d]pyrimidin-2(3H)-one as a base. The modified bases and nucleotides described above are also described in U.S. Patent No. 5,912,340 (granted to Kutyavin et al. on June 15, 1999), the description of which is incorporated herein by reference. The hybridization properties of d2-amA and d2-sT are described in Kutyavin et al. (1996) Biochemistry 35:11170-76, the description of which is also incorporated herein by reference. The synthesis and hybridization properties of d / I and dP are described in Woo et al. (1996) Nucleic Acids Research 25(13):2470-75, the description of which is also incorporated herein by reference. Further examples of G* and C* include 7-alkyl-7-denitroguanine and N, respectively. 4 -alkylcytosine (where the alkyl group is methyl or ethyl), described in Lahoud et al. (2008) Nucleic Acids Research 36(10):3409-19 (the description of which is hereby incorporated by reference). Other examples of G* and C* include 7-nitro-7-denitroxanthine (NitrocH) and 2-thiocytosine (sC), described in Lahoud et al. (2008) Nucleic Acids Research 36(22):6999-7008 (the description of which is hereby incorporated by reference). Hoshinka et al. (2010) Angew Chem Int Ed Engl.49(32):5554-5557 describes the use of such bases (“Self-Avoiding Molecular Recognition Systems”), including 2'-hypoxanthine as G* (the description of which is hereby incorporated by reference; see also: see FIG. 1 See also Yang et al. (2015) Chembiochem. 16(9): 1365-1367 (the description of this reference is hereby incorporated by reference; see especially scheme 1).

[0356]

[0357] In the method described herein for identifying up to forty (or more) target nucleic acids in biological samples collected from subjects, each of the forward primers, reverse primers, and probes in a primer set of a target nucleic acid has less than 10% homology with the forward primers, reverse primers, and probes in different primer sets.

[0358] A method for identifying up to forty (or more) target nucleic acids in a biological sample collected from a subject also includes placing the nucleic acids, primers, and probes within a reaction vessel under amplification conditions, optionally followed by melting curve determination, and detecting the presence of any (multiple) amplification products via real-time PCR and optional melting curve analysis using an optical assembly. A multichannel optical detection assembly is described in US 6,369,893B1, embodiments of which are incorporated herein by reference. The optical assembly utilized in this disclosure preferably comprises at least ten optical channels configured to simultaneously transmit excitation beams in at least ten excitation wavelength ranges and simultaneously detect emitted light to and from at least ten emission wavelength ranges from the reaction vessel. Since the optical assembly may be limited by the number of emission or detection wavelengths, when identifying more than 10 target nucleic acids, one or more probes for detection via melting curve analysis and one or more probes for detection via real-time PCR can be combined in a single optical channel. In some embodiments, the optical components simultaneously detect up to ten (10) emission wavelength ranges in each amplification cycle via real-time PCR in each reaction chamber, and simultaneously detect up to ten (10) emission wavelength ranges in each melting curve cycle. In some embodiments, the method differentially identifies up to thirty (30) target nucleic acids in each melting curve assay by combining probes for multiple (up to three) target nucleic acids in each optical channel and defining a melting window for each target nucleic acid. When two or more probes are combined in an optical channel, the probes detected by melting curve assay are configured with a Tm below the annealing temperature (no amplification curve). However, some channels have multiple probes (up to four probes), but they are either all amplification probes or all melting probes. When more than ten (10) target nucleic acids are to be identified, target nucleic acids from viruses can be detected via real-time PCR, and target nucleic acids from bacteria can be detected via melting curve assay. However, there are exceptions where viruses are detected by melting and bacteria by real-time PCR.

[0359] Methods for identifying up to forty target nucleic acids in biological samples collected from subjects also include differentiating the presence of (multiple) target nucleic acids in a sample based on the detection or absence of (multiple) amplification products, or determining that no target nucleic acids are detectable.

[0360] The lysis chamber, together with the lysis reagents used in the methods described herein, is suitable for lysing viruses, bacteria, fungi, protozoa, and epithelial cells present in biological samples. In some aspects, the methods do not include mechanical lysis of biological samples. For example, the methods may utilize only chemical lysis. In other aspects, the methods include mechanical lysis of biological samples. Biological samples may be selected from respiratory samples (e.g., nasopharyngeal swabs (NP), oropharyngeal swabs (OP), nasal swabs (NS), respiratory mucus samples, respiratory tissue samples, respiratory cell samples), saliva samples, sputum samples, blood, plasma, serum, semen, cerebrospinal fluid, tissue, tears, urine, feces, smear samples, vaginal swabs, vaginal mucus samples, vaginal tissue samples, vaginal cell samples, bacterial cultures, mammalian cell cultures, viral cultures, human cells, bacteria, extracellular fluid, pancreatic fluid, cell lysates, PCR reaction mixtures, in vitro nucleic acid modification reaction mixtures, or combinations thereof. In some instances, the target nucleic acid is selected from viral pathogens, bacterial pathogens, fungal pathogens, protozoan pathogens, or combinations thereof. For example, the target nucleic acid can be selected from viral respiratory pathogens, bacterial respiratory pathogens, or combinations thereof.

[0361] Also disclosed is a sample cartridge for identifying up to forty target nucleic acids in biological samples collected from subjects. The sample cartridge may comprise a housing containing multiple chambers. As described herein, the multiple chambers include a sample chamber having at least one fluid outlet in fluid communication with another of the multiple chambers; a lysis chamber in fluid communication with the sample chamber, wherein the lysis chamber is adapted to perform mechanical and / or chemical lysis to release nucleic acids from the biological sample, optionally wherein the sample chamber and the lysis chamber are the same chamber; a lyophilized reagent chamber containing a lyophilized PCR master mixture comprising at least one primer set for amplifying and detecting target nucleic acids; and one or more liquid reagent chambers containing lysis buffer, binding reagent, and elution buffer. The sample cartridge also includes a reaction vessel fluidly connected to the multiple chambers of the housing and configured for nucleic acid amplification and melting curve determination, and adapted to detect one or more amplified products via real-time PCR and melting curve analysis using optical components. In some embodiments, the reaction vessel may contain up to five reaction chambers, each configured to amplify up to forty target nucleic acids in solution. In some embodiments, the reaction vessel contains a single reaction chamber. The optical assembly contains at least ten optical channels configured to simultaneously transmit excitation beams in at least ten excitation wavelength ranges to the reaction vessel and simultaneously detect emission light in at least ten emission wavelength ranges from the reaction vessel. When identifying more than 10 target nucleic acids, one or more probes for detection via melting curve analysis and one or more probes for detection via real-time PCR are combined in a single optical channel. The sample cartridge also contains a filter disposed in the lysis chamber (if present) or in the fluid path between the sample chamber and the reaction vessel.

[0362] The sample cassette can be further adapted for high-resolution melting detection, nested PCR, multiphase detection, or combinations thereof. In some embodiments, the methods disclosed herein do not include nested PCR.

[0363] A diagnostic assay system for identifying up to forty target nucleic acids in biological samples collected from subjects is also disclosed. The system may include a sample cartridge as described herein; a thermal assembly configured to place nucleic acids, primers, and probes within a reaction vessel of the sample chamber under amplification conditions, optionally followed by melting curve determination; an optical assembly comprising at least ten optical channels and configured to simultaneously transmit excitation beams in at least ten excitation wavelength ranges and simultaneously detect emission light, respectively, in at least ten emission wavelength ranges to and from the reaction vessel; and electronic equipment for (i) receiving and processing signals generated by the optical assembly via real-time PCR and melting curve determination due to the presence of any (multiple) amplification products; and (ii) based on the (multiple) The system includes the detection of amplification products, melting curves, or their absence; differential identification of the presence of target nucleic acids in a sample; or determination of the absence of detectable target nucleic acids; and (iii) outputting real-time PCR results of samples in a reaction vessel during or after amplification, and outputting melting results of samples in a reaction vessel during or after melting probe assays; and a communication interface for communicating with an electronic device comprising a display screen with a user interface displaying one or more graphical elements, allowing the user to input selections corresponding to at least one of a plurality of target assays designed to determine the presence, absence, or amount of at least one target nucleic acid in a biological sample. In some aspects, the system is configured to adjust the number of amplification cycles based on the titer of the target nucleic acid. The respiratory tract assay may be a qualitative test, in which a positive or negative interpretation is given. All targets are preferably tested at high concentrations to ensure they are detected.

[0364] This disclosure considers any method capable of receiving, processing, and transmitting information back and forth between the laboratory conducting the assay, the information provider, the medical personnel, and the subject. For example, in some embodiments of the invention, a sample is obtained from the subject and submitted to a testing service (e.g., a clinical laboratory in a medical institution, a genome mapping company, etc.) located anywhere in the world (e.g., a country different from the country where the subject resides or where the information is ultimately used) to generate raw data. Where the sample comprises tissue or other biological samples, the subject may visit a medical center to collect the sample and deliver it to the testing service, or the subject may collect the sample themselves and deliver it directly to the testing service. Where the sample contains previously identified biological information, the subject may send that information directly to the testing service (e.g., by scanning an information card containing that information with a computer and transmitting the data to a computer at a mapping center using an electronic communication system). Once the testing service receives the sample, it processes it and generates a set of test results specifically tailored to the diagnostic or prognostic information required by the subject.

[0365] Test results can be prepared in a format suitable for interpretation by the attending clinician. For example, the prepared format may represent a diagnosis or risk assessment of the subject, rather than providing raw data, with or without recommendations for specific treatment options. Test results can be displayed to the clinician using any suitable method. For example, in some implementations, the testing service provider generates a report, which can be printed for the clinician (e.g., during on-site consultation) or displayed to the clinician on a computer monitor.

[0366] In some implementations, information is first analyzed at the field of care or at a regional facility. The raw data is then sent to a central processing facility for further analysis and / or to be transformed into information useful to clinicians or patients. Central processing facilities offer advantages such as confidentiality (all data is stored in a central facility with uniform security protocols), speed, and consistency of data analysis. After a subject's treatment, the central processing facility can control the whereabouts of the data. For example, using electronic communication systems, the central facility can provide the data to clinicians, subjects, or researchers.

[0367] In some implementations, participants can directly access data using electronic communication systems. Participants can then choose further interventions or consultations based on the results. In some implementations, the data is used for research purposes. For example, the data can be used to further optimize the inclusion or exclusion of biomarkers, as a useful indicator of a specific symptom or stage of disease, or as a companion diagnostic to determine the course of treatment action.

[0368] Kit

[0369] A kit for performing the methods described herein is also considered. Such a kit includes one or more reagents useful for practicing any of these methods. The kit typically includes packaging having one or more containers holding reagents, either as one or more separate compositions or, optionally, as a mixture where reagent compatibility permits. The kit may also include (multiple) other materials that may be necessary from the user's perspective, such as (multiple) buffers, (multiple) diluents, (multiple) standards, and / or any other materials useful in any other step of sample preparation, washing, or determination.

[0370] The kit preferably includes instructions for performing one or more screening methods described herein. These instructions may be affixed to the packaging material or included as part of the package instructions. While instructions are typically written or printed materials, they are not limited to these. Any medium capable of storing and communicating such instructions to the end user may be used. Such media include, but are not limited to, electronic storage media (e.g., disks, magnetic tapes, cases, chips), optical media (e.g., CD-ROMs), etc. As used herein, the term "instructions" may include the website address providing the instructions.

[0371] In some embodiments, the kit includes primer pairs for amplifying and / or detecting the combined targets of the respiratory pathogen biomarkers described above, optionally including probes specific to these targets. In some embodiments, these kits may include primer pairs and optional probes for detecting one or more of the controls described above.

[0372] In some implementations, the kit may include one or more... Boxes are provided together or in one or more Any of the reagents described above provided in the kit. See, for example, U.S. Patents 5,958,349, 6,403,037, 6,440,725, 6,783,736, and 6,818,185; the description of each of these patents in relation to the present invention is incorporated herein by reference.

[0373] In some implementations, any of the kits described herein may include a container for nasal aspirate / washing samples and / or a swab for collecting nasopharyngeal swab samples. Example

[0374] Example 1: Respiratory Panel Limit of Detection (LOD) Study Report

[0375] This example demonstrates the results of a study on the limit of detection (LOD) of the respiratory tract combination test. The collected data were used to validate the analytical performance of the respiratory tract combination test.

[0376] Abbreviations

[0377] ADF - Assay Definition; CCU - Color Change Unit; CEID - Chicken Embryo Infection Dose; CFU - Colony Forming Unit; CI - Confidence Interval; CLSI - Clinical and Laboratory Standards Institute; Cp - Copy Number; Ct - Cyclic Threshold; CV - Coefficient of Variation; EDTA - Ethylenediaminetetraacetic Acid; FDA - Food and Drug Administration; Flu - Influenza; GX - hMPV - Human metapneumovirus; IRR - International Reagent Resource; IVD - In Vitro Diagnostics; LOD - Limit of Detection; LSP - Batch Specific Parameter; NIBSC - National Institute for Biological Standards and Control; NP - Nasopharyngeal cavity; PFU - Plaque Forming Unit; RNA - Ribonucleic Acid; RSV - Respiratory Syncytial Virus; SD - Standard Deviation; TCID - Tissue Culture Infectious Dose; UTM - Universal Transport Medium; WHO - World Health Organization

[0378] SUMMARY

[0379] Studies were conducted to determine the analytical sensitivity or limit of detection (LOD) for each biological target included in the respiratory tract combination test. The LOD was estimated using probabilistic unit analysis and subsequently validated for each biological target. All respiratory tract combination organisms, strains, and LOD validation levels are presented in Table 1. The test met the Xpert respiratory tract combination LOD acceptance criteria.

[0380] Table 1: Respiratory tract assemblages, bacterial (viral) strains, and validated LODs

[0381]

[0382] *Titer values ​​for clinical samples of coronavirus HKU1 are provided in cp / mL.

[0383] OBJECTIVE

[0384] Determine the analytical sensitivity or limit of detection (LOD) for the respiratory combination test. LOD is defined as the lowest concentration of an analyte that can be consistently detected in ≥95% of the tested samples under routine clinical laboratory conditions and in a defined sample type.

[0385] Materials and Methods

[0386] This study was conducted according to the Xpert respiratory combination limit of detection (LOD) study protocol. The design of this study followed the Clinical and Laboratory Standards Institute (CLSI) Guideline 2012 EP17-A2, except that, in accordance with the communication with the FDA (Q202166), the number of replicates used was less than the number recommended in logistic regression analysis.

[0387] The Level of Observation (LOD) for each target organism was estimated using at least one strain of bacteria or viruses. Additionally, strains representing adenovirus types B, C, and E; all known serotypes of coronaviruses (NL63, HKU1 229E, and OC43); parainfluenza subtypes 1–4; two influenza A strains (H3N2 and H1N1); and two influenza B strains (Yamagata and Victoria lineages) were included in this study. LOD was estimated using logistic regression analysis based on probabilistic units.

[0388] For LOD validation, a concentration with a positive reported result of 95% or higher is defined as a validated LOD.

[0389] Samples

[0390] All viral and bacterial stock solutions were purchased from external suppliers and were titrated cultures, except for the CoronaHKU1 strain, which was a clinical sample whose titer was determined by droplet digital PCR (ddPCR) and the SARS-CoV-2 WHO first international standard (NIBSC 20 / 146). Additionally, some purchased cultures were also titrated by ddPCR to obtain a Level of Displacement (LOD) in cp / mL, required for subsequent design validation studies. Dilution combinations for each bacterial (viral) strain were prepared by adding the virus or bacteria, respectively, to a mixed negative clinical nasopharyngeal (NP) sample matrix at concentrations covering the expected LOD. The goal of preparing serial dilutions was to have at least three levels resulting in a positive detection rate ranging from 0.10 to 0.90, with at least one level exceeding 0.95. The hit rate was defined as the number of replicates with a "detected" result divided by the total number of replicates in the test. A negative sample level was also included, consisting of a clinical sample matrix without the addition of virus or bacteria.

[0391] Test Steps

[0392] The LOD for each organism strain was initially estimated by testing dilution combinations of at least five members covering the expected LOD in a clinical sample matrix. Each member of the combination was tested using two Xpert respiratory combination reagent batches for a minimum of three days, with at least three replicates per day for each batch. Additionally, negative samples were tested after three days, with three replicates per day for each batch. The results of the negative replicates were used for probabilistic unit analysis of all tested organism strains. For each single strain, reagent batch, and combined two reagent batches, the LOD was estimated using logistic regression analysis with probabilistic unit methods.

[0393] Based on the LOD estimate for each bacterial / viral strain, the maximum observed LOD level from different reagent batches was selected for validation. Each bacterial / viral strain was diluted to the selected LOD level for validation in a negative clinical sample matrix. LOD validation was performed using a single Xpert respiratory combination reagent batch with at least 20 replicates, according to CLSI EP17-A2 and the communication with the FDA (Q202166). If validation was unsuccessful (i.e., a positive reporting rate of less than 95%), the step was repeated at a higher concentration. The concentration with a positive reporting rate greater than or equal to 95% (detected in 19 out of 20 replicates) was defined as the validated LOD.

[0394] For each day of testing, an external control was tested during the study period (changing one of the two positive controls).

[0395] External Controls

[0396] Two different external positive controls were used and tested daily using the Xpert respiratory tract combination test. These two external controls consisted of a mixture of target organisms diluted in a serum protein matrix. An external control test was considered valid if all target organisms included in the control mixture were reported as "POSITIVE" and any excluded target organisms were reported as "NEGATIVE". The external control groups, along with the expected valid results, are presented in Table 2.

[0397] Table 2: Composition and Requirements of External Positive Controls

[0398]

[0399] *The pandemic influenza A, H1N1-09, included in external control sample mixture 2 will not only result in a positive report of influenza A H1N1, but also in a positive report of influenza A H3N2. Therefore, positive reports of influenza A H3N2 from external control sample mixtures 1 and 2 are considered valid results.

[0400] Instruments

[0401] All tests were performed using validated software 6.4 in a calibrated environment. Performed on the instrument system.

[0402] Replicates

[0403] If the initial Xpert respiratory combination test is reported as indeterminate (INVALID, ERROR, NO RESULT), a single retest should be performed, provided there are sufficient samples available for retesting. If the retest result is still indeterminate, report accordingly.

[0404] Data Analysis

[0405] use The probability unit analysis in version 19.2020.1 estimates the Level of Detection (LOD) for each target organism using logistic regression analysis. Detection rates at each dilution are converted to normal probability units and plotted relative to their nominal concentrations, then fitted using a regression model. The LOD is estimated using the concentration of virus / bacteria at which the detection rate equals 95% as determined by logistic regression analysis. The LOD is calculated for each individual bacterial (viral) strain for each reagent batch and for two combined reagent batches. The highest observed LOD from the analyses of different reagent batches is selected for subsequent validation.

[0406] LOD validation is performed by calculating the positivity rate of each target organism strain. For LOD to be considered valid, each analyte needs to be consistently detected in ≥95% of the test samples.

[0407] Table 3 defines the expected analytical sensitivity / LOD for each target organism in the Xpert respiratory tract combination values.

[0408] Table 3: Xpert's Expected LOD for the Airway

[0409] Target Organisms Expected LoD Adenovirus < 1000 TCID50 / mL Coronavirus < 250 TCID50 / mL SARS-CoV-2 < 250 Cp / mL Human Metapneumovirus (hMPV) < 1000 TCID50 / mL Rhinovirus / Enterovirus < 100 TCID50 / mL Parainfluenza < 100 TCID50 / mL Influenza A (Flu A) < 1000 TCID50 / mL Influenza A (Flu A) H1N1pdm < 1000 TCID50 / mL Influenza B (Flu B) < 1000 TCID50 / mL Respiratory Syncytial Virus (RSV) < 5 TCID50 / mL Bordetella pertussis < 300 CFU / mL Bordetella parapertussis < 300 CFU / mL Mycoplasma pneumoniae < 100 CCU / mL Chlamydia pneumoniae < 100 TCID50 / mL

[0410] *Using the first WHO international standard (20 / 146) to provide LOD for SARS-CoV-2

[0411] Results and Analysis

[0412] LOD Verification

[0413] The LOD of each Xpert respiratory combination organism, estimated by probability units at 95% CI, is presented in Table 4.

[0414] Table 4: Probability Unit Results (95% CI for each Xpert respiratory tract combination target organism, detailed for each reagent batch and for combined two reagent batches)

[0415]

[0416]

[0417]

[0418] *The number of positive detections in the range of 0.1–0.9 was less than three, therefore no statistical results are presented; see Section 7.2.

[0419] Reported False Results

[0420] Of the 3,690 test boxes, 22 provided error reporting results, representing 0.6%. The overall error rate was below the standard of 1.4%.

[0421] External Controls

[0422] All external controls passed the acceptance criteria.

[0423] CONCLUSIONS

[0424] The Level of Detection (LOD) of Xpert respiratory combination target organisms was estimated using probabilistic unit analysis and validated at a level where each analyte was consistently detected at a rate of ≥95% of the tested samples. The empirical LOD for each Xpert respiratory combination target organism was below the values ​​defined in Table 3, thus meeting the acceptance criteria.

[0425] Example 2: Cross Talk

[0426] Crosstalk is an unwanted optical signal present in a channel, caused by and directly proportional to the optical signals in adjacent channels. Crosstalk can be positive or negative, and crosstalk from different adjacent channels is additive. Preliminary crosstalk factors were determined based on amplification and melting curves (data not shown).

[0427] A simplified crosstalk study was conducted to obtain preliminary crosstalk factors for the amplification and melting curves. Slight melting crosstalk was observed from TxR to Alx532 (data not shown). Apart from this, melting crosstalk was only detected to the non-melting channels. Therefore, a melting crosstalk factor was not set at this stage because results in the non-melting channels do not affect the output, and the melting channels are separated in TxR & Alx532, thus not leading to erroneous results.

[0428] The final major crosstalk factor of the amplification and melting curves was evaluated. Additionally, a preliminary residual crosstalk factor was defined.

[0429] Example 3: Respiratory panel cut-off evaluation

[0430] Objectives

[0431] Evaluate changes to the assay definition document (ADF) settings and their impact on analytical, clinical, and manufacturing practices.

[0432] Initial results from the LOD study indicate that respiratory combination assays can report positive results in the high Ct / low titer range. Delayed positive interpretations arise from LOD levels below the pathogen, failure to detect by comparative assays, environmental contamination, or incidental nonspecific amplification.

[0433] The following changes were implemented to ensure manufacturability by improving the robustness of the assay kit during manufacturing and operation. Changes were also implemented to further reduce the risk of false positives due to potential, unforeseen oligonucleotide interactions or other nonspecific amplifications. Additionally, optical settings were adjusted to reduce the risk of unnecessary invalid readings due to curve fitting bias and crosstalk.

[0434] Supporting data

[0435] To evaluate potential setup changes, data from Phase II preclinical studies, preliminary kit stability, development batch data, and analytical studies were reviewed for each target (if applicable). Setup changes are designed to increase tolerability for positive reads due to environmental contamination and nonspecific amplification, without significantly impacting clinical performance, kit shelf life, or analytical sensitivity. Setup changes also aim to reduce unnecessary invalid reads.

[0436] Changes in SARS-CoV-2 effective maximum cycle number

[0437] In the preclinical phase II study, eight SARS-CoV-2 positive samples were detected using a respiratory tract assay; however, comparative assays reported negative results, thus classifying them as false positives. In the design validation study data (collected up to the time of design change), four replicates with unexpected SARS-CoV-2 positive interpretations were observed.

[0438] By reducing the maximum effective number of cycles from 40 to 39, the number of false positives in the design validation study will be reduced by 3. In the preclinical study, one additional sample was assessed as a false negative.

[0439] Preliminary kit stability data for the latest technical feasibility batch were reviewed. For kits stored at 50°C, the new maximum effective number of cycles resulted in three replicates being reported as negative. For kits stored at 4°C, 28°C, and 35°C, the Ct values ​​at all test time points were significantly lower than the new maximum effective number of cycles, and no change was reported.

[0440] PQC data of SARS-CoV-2 in research and development batches are as follows Figure 8 The new effective maximum number of cycles does not change any reported results for the SARS-CoV-2 control material, and the mean Ct is unaffected.

[0441] Changes in influenza A (Flu A) maximum effective cycle number

[0442] In a preclinical phase II study, a Flu A-positive sample was reported using a respiratory tract assay, but was reported negative using a comparative assay, and was therefore classified as a false positive. In the data from the design validation study (collected up to the design change), one replicate with an unexpected Flu A-positive interpretation was observed. The Ct / EPF value for this replicate was 41.7 / 70.

[0443] By reducing the maximum effective cycle number for Flu A from 45 to 40, both samples with false positive interpretations were reported as Flu A negative. In the preclinical phase II study, reducing the maximum effective cycle number did not produce any false negative results because the Ct values ​​for all true positive samples were below 40.

[0444] PQC data for the R&D batches of FluA and FluA H1N1-09 are as follows: Figure 9 The new effective maximum number of cycles does not change any reported results for the Flu A control material and Flu AH1N1-09, and the average Ct is unaffected.

[0445] Changes in RSV manual threshold fluorescence units

[0446] In the preclinical phase II study, three RSV-positive samples (two clinical samples and one design sample) were detected using a respiratory tract assay; however, they were reported as negative using a comparative assay and were therefore classified as false positives. During design validation, a total of six replicates yielded unexpected RSV-positive interpretations. By raising the threshold to 50, the one false positive (clinical sample) in the preclinical phase II study and the five replicates with unexpected positive results in design validation will all be reported as RSV-negative.

[0447] Increasing the manual threshold fluorescence will result in a Ct shift. Reanalyze the preliminary kit stability data with the increased threshold and the shifted Ct. Evaluate thresholds of 35 and 50 and compare them to the current threshold of 20. In the preliminary kit stability study, a shifted Ct value with the evaluated threshold will not change any reported results. At time point zero (T=0), a Ct of approximately 33–34 will be obtained with a threshold of 50.

[0448] PQC data of R&D batches of RSV as follows Figure 10 The new manual threshold does not change any reported results for the RSV-A control material and RSV-B.

[0449] Changes in rhinovirus effective maximum cycle number, melt peak threshold, melt valley threshold, and melt window

[0450] Detection of rhino-enterovirus targets is performed by amplification and / or melting. By reducing the maximum effective cycle number from 40 to 39 and by adjusting the melting settings (melting peak threshold from 2 to 6 and melting trough from -2 to -4), several false positive reads obtained during design validation will be reported as rhino-enterovirus negative.

[0451] In the preliminary kit stability study, a Rhino strain was included, primarily detected by amplification. By reducing the maximum effective cycle number from 40 to 39, the reported result of an abnormal replicate at time point zero (T=0) changed from positive to negative. All other data points were below the new maximum effective cycle number and therefore remained positive.

[0452] Enteroviruses are primarily detected via melting curves. Preliminary inclusion studies have identified Coxsackieviruses as having weaker melting curves compared to other enterovirus strains. To evaluate the impact of the new setting, three Coxsackievirus strains were diluted to near the level of limit of observation (LOD), and the data were analyzed using the new melting peak and trough thresholds. Coxsackievirus strains A21 and A9, being desired inclusion strains, were reported positive at the LOD level with the updated melting setting. Coxsackievirus strain B5 (a non-desirable inclusion strain) may have a slightly higher LOD at the updated setting.

[0453] By reducing the maximum temperature of the melting window from 75°C to 65°C, the risk of misinterpretation due to erroneous melting curves will be reduced. For T m All melting curves above 65°C will be used for rhino-enterovirus Ct detection, in addition to the melting curves, and therefore a positive interpretation will be based on amplification.

[0454] Similarly, as with the preliminary kit stability studies, a Rhino strain was included in the testing. The PQC data for the development batch of Rhino are as follows: Figure 11 The new effective maximum cycle number does not change any reported results for the rhinovirus control material, and the average Ct is unaffected. The melting setting does not affect the data because the rhinovirus control material is detected by amplification.

[0455] Changes in mycoplasma (Mycoplasma pneumoniae) melt peak threshold and melt valley threshold

[0456] For Mycoplasma pneumoniae, background noise close to the melting peak and trough thresholds was observed. To reduce the risk of false positives due to background noise, the thresholds were increased. In preclinical Phase II, no samples were assessed as false positives or false negatives compared to the comparative method. By increasing the melting peak threshold from 2 to 5 and decreasing the melting trough threshold from -2 to -4, the reported results for the samples remained unchanged. All Mycoplasma pneumoniae-positive samples will still be reported as positive.

[0457] In the data from the design validation study (collected up to the time of design change), a total of 6 replicates were observed to have unexpected positive interpretations for Mycoplasma pneumoniae. By adjusting the melting peak threshold (from 2 to 5, and the melting trough from -2 to -4), 2 of these replicates will be reported as negative for Mycoplasma pneumoniae.

[0458] The PQC data for the Myco R&D batch is shown in Figure 12. The new melting peak threshold does not change any reported results for the Myco control material, and the melting peak height and valley value are unaffected.

[0459] Changes in Bordetella pertussis melt peak threshold and melt valley threshold

[0460] In the preclinical Phase II study, a total of 12 Bordetella pertussis false-positive samples were detected compared to Biofire. In the design validation study data (collected up to the time of design change), 3 replicates with unexpected Bordetella pertussis positive interpretations were observed.

[0461] By adjusting the melting settings (melting peak threshold from 4 to 7.5, melting trough from -3 to -7), the number of false positives in the preclinical Phase II study will be reduced by 4, and the number of false positives in the design validation study will be reduced by 2. In the preclinical Phase II study, one additional sample will be assessed as a false negative.

[0462] The PQC data for the research batch of Bordetella pertussis are shown in Figure 13. The new melting peak threshold does not change any reported results for the Bordetella pertussis control material, and the melting peak height and trough value are not affected.

[0463] Coronavirus and adenovirus curve fitting

[0464] During the LOD study, crosstalk was observed from FAM-2 (SPC detection) to CF7 (adenovirus) and CF9 (coronavirus) causing shape variation curves in true positive samples with low levels of adenovirus and coronavirus. These shape variation curves did not conform to the curve fitting settings (shape and NRSE criteria) and were reported as invalid runs. The likelihood of these crosstalk issues occurring was assessed as high because FAM-2 (SPC) always has a positive readability, potentially affecting the results for CF7 and CF9. To reduce the risk of shape or NRSE errors in samples at the LOD level, the curve fitting settings for adenovirus and coronavirus were modified.

[0465] Cross-talk from CF3 (Alx532) to FAM-2 factor

[0466] Crosstalk studies conducted during the technical feasibility study concluded that the average crosstalk was -0.3% from CF3 to FAM-2. According to the Procedure for Measuring Assay - Specific Crosstalk Correction Factors, crosstalk values ​​<0.5% can be rounded to zero, which was done in the original crosstalk factor settings.

[0467] A follow-up study conducted during the design and development phase revealed similar crosstalk. However, because CF3 typically exhibits a strong signal, even a low percentage of crosstalk can have a significant impact in the FAM-2 channel, posing a risk of generating background errors. To mitigate this risk, a -0.5% crosstalk factor was added from CF3 to FAM. This added negative crosstalk factor does not implicate the risk of generating false positives in FAM-2, as this channel detects SPCs, which should always be positive.

[0468] Residual cross-talk

[0469] The impact of residual crosstalk factors was evaluated for all data generated to date. Residual crosstalk factors with evidence of miscorrection or lack of necessary correction were removed. Removing non-contributing factors reduces the risk of miscorrection, which could otherwise lead to inaccurate results.

[0470] Impact of changes

[0471] Impact on analysis performance: The data generated so far were reanalyzed, and the analysis LOD was determined based on the updated settings. The reanalysis of the data was considered sufficient because the update of the ADF settings only affected post-run analysis of the data. Therefore, no further study was necessary.

[0472] Based on the altered reporting results, new PPA and NPA were calculated for all targets. Table 5 reflects the performance before and after the changes in the preclinical phase II study.

[0473] Table 5: Preclinical Phase II PPA and NPA before and after the change

[0474]

Claims

1. A kit for detecting and / or identifying viral and bacterial respiratory pathogens in a sample, the kit comprising: A housing containing multiple chambers, wherein the multiple chambers include: A sample chamber, the sample chamber having at least one fluid outlet in fluid communication with another of the plurality of chambers; as well as An optional pyrolysis chamber in fluid communication with the sample chamber, wherein the sample chamber and the pyrolysis chamber are the same chamber; A reaction vessel, fluidly connected to multiple chambers of the housing, and configured for: i) amplification of nucleic acids and ii) detection and identification of multiple amplification products via real-time PCR, melting curve analysis, or a combination thereof; A filter, provided in the fluid path between the pyrolysis chamber (if present) or the sample chamber and the reaction vessel, and A set of primers and optional probes for detecting and / or identifying the presence of alpha-coronavirus, beta-coronavirus, SARS-CoV-2, adenovirus, Chlamydia pneumoniae, influenza A, influenza B, metapneumovirus, rhinovirus / enterovirus, mycoplasma, Bordetella spp., parainfluenza, and respiratory syncytial virus (RSV).

2. The box of claim 1, wherein the lysis chamber contains one or more lysis reagents for releasing nucleic acids.

3. The cartridge of claim 1 or claim 2, wherein the reaction vessel comprises up to four reaction chambers for detecting multiple amplification products in solution.

4. The cartridge of any one of claims 1-3, wherein the reaction container comprises a reaction chamber for multiplex amplification and detection of multiple amplification products in solution.

5. The kit according to any one of claims 1-4, wherein the detection and identification of multiple amplification products are performed via real-time PCR and melting curve analysis.

6. The kit of claim 3 or claim 4, wherein each reaction chamber is configured to simultaneously amplify and detect at least 10 amplification products via real-time PCR.

7. The box of claim 3 or claim 4, wherein each reaction chamber is configured to simultaneously detect up to 10 amplification products via melting curve analysis.

8. A method for detecting and / or identifying viral and bacterial respiratory pathogens in a sample, the method comprising: a) Contact the nucleic acid from the sample with a set of primers and optional probes for detecting the presence of α-coronavirus, β-coronavirus, SARS-CoV-2, adenovirus, Chlamydia pneumoniae, influenza A, influenza B, metapneumovirus, rhinovirus / enterovirus, mycoplasma, Bordetella spp., parainfluenza, and respiratory syncytial virus (RSV). b) Place the nucleic acid, primers, and optional probe under amplification conditions, followed by melting curve determination; c) Detection of the presence of any (multiple) amplification products via real-time PCR and melting curve analysis; as well as d) Based on the detection or absence of the (multiple) amplification products, respectively, identify the presence of viral and / or bacterial respiratory pathogens in the sample, or determine the absence of viral or bacterial pathogens detectable using the primer set.

9. The method of claim 8, wherein the method further comprises administering a treatment regimen to the subject based on the detection of the presence of at least one of the viral or bacterial respiratory pathogens and / or the severity of the infection.

10. The method of claim 9, wherein the treatment regimen is selected from one or more of antibiotic administration, antiviral drug administration, mechanical ventilation, invasive monitoring, sedation, admission to intensive care, surgical intervention, last resort pharmacological treatment, or hospitalization.

11. The method of any one of claims 8-10, wherein the bacterial respiratory pathogen is identified by melting curve analysis, and the viral respiratory pathogen is identified by real-time PCR and / or melting curve analysis.

12. The method of any one of claims 8-10, wherein the viral and / or bacterial respiratory pathogens identified by melting curve analysis include metapneumovirus, influenza A, Mycoplasma pneumoniae, Chlamydia pneumoniae, Bordetella parapertussis, and Bordetella pertussis.

13. The method according to any one of claims 8-12, wherein: a) The contacting of the nucleic acid from the sample with the primers and optional probes of the group comprises: The sample is placed in a box comprising a box body having multiple fluidly connected chambers, a reaction container having one or more reaction chambers and configured for amplifying and detecting the nucleic acid, a fluid path between the multiple chambers and the reaction container, and a filter in the fluid path; as well as If the sample contains cells, the cells in the sample are lysed using one or more lysis reagents present in at least one of the plurality of chambers; (b) The description of placing the nucleic acid, primer pair, and optional probe under amplification conditions includes amplifying the nucleic acid using primers and probes present in a solution within the reaction vessel.

14. The method of claim 13, wherein the detection is performed in a solution in up to four reaction chambers.

15. The method of claim 13, wherein the detection is performed in a solution in a single reaction chamber.

16. The method of any one of claims 8-15, wherein the sample is a respiratory sample selected from nasopharyngeal swabs (NP), oropharyngeal swabs (OP), nasal swabs (NS), respiratory mucus samples, respiratory tissue samples, respiratory cell samples, saliva samples, sputum samples, or combinations thereof.

17. The method of any one of claims 8-15, wherein the sample is a wastewater sample.

18. A reagent kit comprising a cartridge, the cartridge comprising: A set of primers and optional probes for detecting the presence of alpha-coronavirus, beta-coronavirus, SARS-CoV-2, adenovirus, Chlamydia pneumoniae, influenza A, influenza B, metapneumovirus, rhinovirus / enterovirus, mycoplasma, Bordetella spp., parainfluenza, RSV, or combinations thereof; and One or more lysis reagents may be selected.

19. The box of any one of claims 1-7, the method of any one of claims 8-17, or the kit of claim 18, wherein: Primers and optional (multiple) probes used to detect alpha coronaviruses are able to hybridize with the spike glycoprotein gene of alpha coronaviruses; Primers and optional (multiple) probes used to detect β-coronavirus are able to hybridize with the ORF1a gene of β-coronavirus type A; Primers and optional (multiple) probes used to detect SARS-CoV-2 coronavirus are capable of hybridizing with the E gene, N gene, RDRP gene, or combinations thereof of SARS-CoV-2 coronavirus; Primers and optional (multiple) probes used for detecting adenovirus can hybridize with the hexagonal protein genes of adenovirus types A, B, C, D, and E. Primers and optional (multiple) probes used for detecting Chlamydia can hybridize with the ompA gene of Chlamydia pneumoniae; Primers and optional (multiple) probes used to detect influenza A can hybridize with the HA gene, MP gene, PA gene, PB gene or combinations thereof of influenza A; Primers and optional (multiple) probes used to detect influenza B can hybridize with the influenza B MP gene, NS gene, or a combination thereof; Primers and optional (multiple) probes used to detect metapneumovirus can hybridize with the N gene of human metapneumovirus. Primers and optional (multiple) probes used for detecting rhinovirus / enterovirus are capable of hybridizing with the 5'UTR gene of rhinovirus / enterovirus. Primers and optional (multiple) probes used to detect mycoplasma can hybridize with the P1 adhesion protein gene of mycoplasma; Primers and optional (multiple) probes for detecting Bordetella spp. include primers and optional (multiple) probes capable of hybridizing with the IS1001 gene of Bordetella parapertussis. Primers and optional (multiple) probes for detecting Bordetella spp. also include primers and optional (multiple) probes capable of hybridizing with the IS481 gene of Bordetella pertussis. Primers and optional probes for detecting parainfluenza can hybridize with the polymerase (L) protein gene, nucleocapsid (NP) protein gene, or combinations thereof, of parainfluenza types 1, 2, 3, and 4; and Primers and optional (multiple) probes used to detect RSV can hybridize with the RSV A gene, RSV B gene, or a combination thereof.

20. The box, method, or kit of claim 19, wherein, When it exists: Primers and optional (multiple) probes capable of selectively hybridizing with the ORF1a gene of CoV-OC43 contain sequences that are identical or complementary to at least 15 consecutive nucleotides of SEQ ID NO: 24; Primers and optional (multiple) probes capable of selectively hybridizing with the ORF1a gene of CoV-HKU1 contain sequences that are identical or complementary to at least 15 consecutive nucleotides of SEQ ID NO: 28; Primers and optional (multiple) probes capable of selectively hybridizing with the S gene of CoV-229E contain sequences that are identical or complementary to at least 15 consecutive nucleotides of SEQ ID NO: 20; Primers and optional (multiple) probes capable of selectively hybridizing with the S gene of CoV-NL63 contain sequences that are identical or complementary to at least 15 consecutive nucleotides of SEQ ID NO: 32; Primers and optional (multiple) probes capable of selectively hybridizing with the N gene of SARS-CoV-2 contain sequences that are identical or complementary to at least 15 consecutive nucleotides of SEQ ID NO: 96; Primers and optional (multiple) probes capable of selectively hybridizing with the RDRP gene of SARS-CoV-2 contain sequences that are identical or complementary to at least 15 consecutive nucleotides of SEQ ID NO: 104; Primers and optional (multiple) probes capable of selectively hybridizing with the E gene of SARS-CoV-2 contain sequences that are identical or complementary to at least 15 consecutive nucleotides of SEQ ID NO: 100; Primers and optional (multiple) probes capable of selectively hybridizing with the hexagonal protein genes of adenovirus types A, B, C, D and E contain sequences that are identical or complementary to at least 15 consecutive nucleotides of SEQ ID NO: 4; Primers and optional (multiple) probes capable of selectively hybridizing with the ompA gene of Chlamydia pneumoniae contain sequences that are identical or complementary to at least 15 consecutive nucleotides of SEQ ID NO: 16; Primers and optional (multiple) probes capable of selectively hybridizing with the PB gene of influenza A contain sequences that are identical or complementary to at least 15 consecutive nucleotides of SEQ ID NO: 40; Primers and optional (multiple) probes capable of selectively hybridizing with the MP gene of influenza A contain sequences that are identical or complementary to at least 15 consecutive nucleotides of SEQ ID NO: 36; Primers and optional (multiple) probes capable of selectively hybridizing with the PA gene of influenza A contain sequences that are identical or complementary to at least 15 consecutive nucleotides of SEQ ID NO: 44; Primers and optional (multiple) probes capable of selectively hybridizing with the H1 gene of influenza A contain sequences that are identical or complementary to at least 15 consecutive nucleotides of SEQ ID NO: 48; Primers and optional (multiple) probes capable of selectively hybridizing with the MP gene of influenza B contain sequences that are identical or complementary to at least 15 consecutive nucleotides of SEQ ID NO: 52; Primers and optional (multiple) probes capable of selectively hybridizing with the NS gene of influenza B contain sequences that are identical or complementary to at least 15 consecutive nucleotides of one or more of SEQ ID NO: 56; Primers and optional (multiple) probes capable of selectively hybridizing with the N gene of human metapneumovirus contain sequences that are identical or complementary to at least 15 consecutive nucleotides of SEQ ID NO: 60; Primers and optional (multiple) probes capable of selectively hybridizing with the 5' UTR gene of rhinovirus / enterovirus contain sequences that are identical or complementary to at least 15 consecutive nucleotides of SEQ ID NO: 84; Primers and optional (multiple) probes capable of selectively hybridizing with the P1 adhesion protein gene of Mycoplasma pneumoniae contain sequences identical or complementary to at least 15 consecutive nucleotides of SEQ ID NO: 64; Primers and optional (multiple) probes capable of selectively hybridizing with the IS1001 gene of Bordetella parapertussis contain sequences identical or complementary to at least 15 consecutive nucleotides of SEQ ID NO:

8. Primers and optional (multiple) probes capable of selectively hybridizing with the IS481 gene of Bordetella pertussis contain sequences identical or complementary to at least 15 consecutive nucleotides of SEQ ID NO: 12; Primers and optional (multiple) probes capable of selectively hybridizing with the polymerase (L) protein genes of parainfluenza types 1 and 3 contain sequences that are identical or complementary to at least 15 consecutive nucleotides, respectively, to one or more of SEQ ID NO: 68 and 76; Primers and optional (multiple) probes capable of selectively hybridizing with the nucleocapsid (NP) protein genes of parainfluenza types 2 and 4 contain sequences that are identical or complementary to at least 15 consecutive nucleotides, respectively, to one or more of SEQ ID NO: 72 and 80; Primers and optional probes capable of selectively hybridizing with the RSV A gene contain a sequence identical or complementary to at least 15 consecutive nucleotides of SEQ ID NO: 88; and Primers and optional (multiple) probes capable of selectively hybridizing with the RSV B gene contain sequences that are identical or complementary to at least 15 consecutive nucleotides of SEQ ID NO:

92.

21. The kit of any one of claims 1-7, the method of any one of claims 8-17, the kit of claim 18, or the kit, method, or kit of claims 19-20, wherein at least one of the primers and optional probes comprises a detectable marker.

22. The box, method, or kit of claim 21, wherein at least one probe comprises a fluorescent dye and a quencher molecule.

23. The kit of any one of claims 1-7, the method of any one of claims 8-17, the kit of claim 18, or the kit, method, or kit of any one of claims 19-22, wherein the kit or kit further comprises primer pairs specific to exogenous controls and / or endogenous controls, or the method further employs primer pairs specific to exogenous controls and / or endogenous controls, wherein the exogenous control is a sample processing control, and wherein the endogenous control is a sample adequacy control.

24. The kit of any one of claims 1-7, the method of any one of claims 8-17, or the kit of claim 18, wherein the kit or kit facilitates the detection of viral and / or bacterial respiratory pathogens in the biological sample within 60 minutes, 45 minutes, or 30 minutes of collection from the subject, and / or the method comprises detecting viral and / or bacterial respiratory pathogens in the biological sample within 60 minutes, 45 minutes, or 30 minutes of collection from the subject.

25. The kit of any one of claims 2-7, the method of any one of claims 13-17, the kit of claim 18, or the kit, method, or kit of any one of claims 19-24, wherein the one or more lysis reagents provide simultaneous lysis of viral and bacterial cells.

26. The kit or method of any one of claims 1-17, wherein the limit of detection (LOD) of the kit or method is greater than or equal to about 25 TCID50 / mL for adenovirus, greater than or equal to about 0.1 TCID50 / mL for coronavirus 229E, greater than or equal to about 100 copies / mL for coronavirus HKU1, greater than or equal to about 0.1 TCID50 / mL for coronavirus NL63, greater than or equal to about 0.1 TCID50 / mL for coronavirus OC43, greater than or equal to about 1500 IU / mL for SARS-CoV-2, greater than or equal to about 0.5 TCID50 / mL for human metapneumovirus (hMPV), greater than or equal to about 4 TCID50 / mL for human rhinovirus, greater than or equal to about 8 TCID50 / mL for enterovirus, greater than or equal to about 8 TCID50 / mL for parainfluenza type 1, and greater than or equal to about 2 TCID50 / mL for parainfluenza type 2. TCID50 / mL is defined as follows: ≥10 TCID50 / mL for parainfluenza serotype 3; ≥8 TCID50 / mL for parainfluenza serotype 4; ≥16 TCID50 / mL for influenza A H3N2; ≥0.5 TCID50 / mL for influenza A H1N1; ≥4.4 TCID50 / mL for influenza B; ≥0.1 TCID50 / mL for respiratory syncytial virus (RSV) A; ≥0.05 TCID50 / mL for RSV B; ≥200 CFU / mL for Bordetella pertussis; ≥8 CFU / mL for Bordetella parapertussis; ≥15 CCU / mL for Mycoplasma pneumoniae; and / or ≥1 CCU / mL for Chlamydia pneumoniae. TCID50 / mL.

27. A system for detecting pathogens in biological samples, the system comprising: Module, the module having a receiving slot for receiving a cartridge according to any one of claims 1-7, wherein the module includes one or more mechanisms within the receiving slot for manipulating a fluid sample within the cartridge, and instruments connected to the interface of the reaction vessel; and A memory on which programmable instructions are recorded, the programmable instructions being specifically configured to operate the module according to a respiratory pathogen assay protocol to determine the nucleic acid sequence characteristics of alpha-coronavirus, beta-coronavirus, SARS-CoV-2, adenovirus, Chlamydia pneumoniae, influenza A, influenza B, metapneumovirus, rhinovirus / enterovirus, mycoplasma, Bordetella spp., parainfluenza, and respiratory syncytial virus (RSV).

28. A method for identifying up to forty target nucleic acids in a biological sample collected from a subject, the method comprising: a. Provide a sample box, the sample box comprising: i. A plurality of chambers, said plurality of chambers comprising: A lysis chamber having at least one fluid outlet in fluid communication with another of the plurality of chambers, the lysis chamber being adapted to perform mechanical and chemical lysis to release nucleic acids from the biological sample; ii. Lyophilized reagents and liquid reagents in two or more of the plurality of chambers, wherein the lyophilized reagents contain at least one set of primers for amplifying and detecting the target nucleic acid, and the liquid reagents include lysis buffer, binding reagent and elution buffer; as well as iii. A reaction vessel, which is fluidly connected to a plurality of chambers of the sample cartridge and configured to amplify the target nucleic acid; b. Contact the nucleic acid from the biological sample with the at least one primer set. i. Each primer set contains a forward primer, a reverse primer, and a probe specific to the target nucleic acid in the biological sample, and ii. At least one of the forward or reverse primers in each group contains one or more modified bases, said modified bases being selected from unstable bases, stable bases, or combinations thereof; as well as iii. Each probe in each group contains a detectable portion for detection via real-time PCR or melting curve analysis; c. Place the nucleic acid, primers, and probes within the reaction vessel under amplification conditions, optionally followed by melting curve determination. d. Detect the presence of any (multiple) amplification products via real-time PCR and optional melting curve analysis using an optical assembly, wherein the optical assembly comprises at least ten optical channels configured to simultaneously transmit excitation beams in at least ten excitation wavelength ranges and simultaneously detect emission light in at least ten emission wavelength ranges to and from the reaction vessel. as well as e. Based on the detection or absence of the (multiple) amplification products, differentially identify the presence of (multiple) target nucleic acids in the sample, or determine that no target nucleic acid is detectable. When identifying more than 10 target nucleic acids, one or more probes for detection via melting curve analysis and one or more probes for detection via real-time PCR are combined in a single optical channel.

29. The method of claim 28, wherein the lysis chamber, together with the lysis reagent, is adapted to lyse viruses, bacteria, fungi, protozoa, and epithelial cells present in the biological sample.

30. The method of claim 29, wherein the method does not include mechanical lysis of the biological sample.

31. The method of claim 29, wherein the method further comprises mechanical lysis of the biological sample.

32. The method of any one of claims 28-31, wherein the biological sample is selected from respiratory samples, the respiratory samples comprising nasopharyngeal swab (NP) samples, oropharyngeal swab (OP) samples, nasal swab (NS) samples, respiratory mucus samples, respiratory tissue samples, or respiratory cell samples; samples comprising saliva, sputum, blood, plasma, serum, semen, cerebrospinal fluid, tissue, tears, urine, feces; or smear preparations, vaginal swab samples, vaginal mucus samples, vaginal tissue samples, vaginal cell samples, bacterial cultures, mammalian cell cultures, viral cultures, human cell samples, bacterial samples, extracellular fluid samples, pancreatic juice samples, cell lysates, PCR reaction mixtures, in vitro nucleic acid modification reaction mixtures, or combinations thereof.

33. The method of any one of claims 28-32, wherein the target nucleic acid is selected from viral pathogens, bacterial pathogens, protozoan pathogens, or combinations thereof.

34. The method of any one of claims 28-32, wherein the optical component simultaneously detects up to ten (10) emission wavelength ranges in each amplification cycle via real-time PCR in each reaction chamber, and simultaneously detects up to ten (10) emission wavelength ranges in each melting curve cycle.

35. The method of claim 34, wherein the method differentially identifies up to thirty (30) target nucleic acids in each melting curve determination by combining probes for multiple (up to three) target nucleic acids in each optical channel and defining a melting window for each target nucleic acid.

36. The method of any one of claims 28-35, wherein when two or more probes are combined in an optical channel, the probes detected by melting curve determination are configured such that Tm is below the annealing temperature.

37. The method of any one of claims 28-36, wherein the unstable base is selected from sterically hindered bases that reduce nonspecific primer interactions, bases that suppress the melting temperature of double-stranded primers, bases that suppress unwanted primer-dimer extension, or combinations thereof.

38. The method of any one of claims 28-37, wherein the stabilizing base increases the melting temperature of the double-stranded primer or reduces secondary structure formation.

39. The method of any one of claims 28-38, wherein the cassette is further adapted to perform high-resolution melting detection, nested PCR, multiphase detection, or a combination thereof.

40. The method of any one of claims 28-39, wherein the method does not include nested PCR.

41. A sample kit for identifying up to forty target nucleic acids in a biological sample collected from a subject, the kit comprising: a. A housing containing multiple chambers, wherein the multiple chambers include: i. A sample chamber having at least one fluid outlet in fluid communication with another of the plurality of chambers; ii. A lysis chamber in fluid communication with the sample chamber, wherein the lysis chamber is adapted to perform mechanical and chemical lysis to release nucleic acids from the biological sample, optionally wherein the sample chamber and the lysis chamber are the same chamber; i. A lyophilized reagent compartment containing a lyophilized PCR master mixture comprising at least one primer set for amplifying and detecting the target nucleic acid, further... a. Each primer set contains a forward primer, a reverse primer, and a probe specific to the target nucleic acid in the biological sample, and b. At least one of the forward or reverse primers in each group contains one or more modified bases, wherein the modified bases are selected from unstable bases, stable bases, or combinations thereof; as well as c. Each group of probes contains a detectable portion that is used for detection via real-time PCR or melting curve analysis; iii. One or more liquid reagent chambers, said liquid reagent chambers containing lysis buffer, binding reagent and elution buffer; b. A reaction vessel, fluidly connected to multiple chambers of the housing, configured for nucleic acid amplification and melting curve determination, and adapted to detect one or more amplification products via real-time PCR and melting curve analysis using optical components. The optical component includes at least ten optical channels configured to simultaneously transmit excitation beams in at least ten excitation wavelength ranges to the reaction vessel and simultaneously detect emitted light in at least ten emission wavelength ranges from the reaction vessel. as well as c. A filter disposed in the fluid path between the pyrolysis chamber and the reaction vessel; and When identifying more than 10 target nucleic acids, one or more probes for detection via melting curve analysis and one or more probes for detection via real-time PCR are combined in a single optical channel.

42. A diagnostic assay system for identifying up to forty target nucleic acids in biological samples collected from a subject, the system comprising: a. The sample box according to claim 93; b. A thermal assembly configured to place the nucleic acids, primers, and probes within the reaction vessel of the sample chamber under amplification conditions, optionally followed by melting curve determination; c. An optical assembly comprising at least ten optical channels and configured to simultaneously transmit excitation beams in at least ten excitation wavelength ranges and simultaneously detect emitted light in at least ten emission wavelength ranges to and from the reaction vessel; d. Electronic equipment used for i. Receive and process signals generated by the optical components in the presence of any (multiple) amplification products as detected by real-time PCR and melting curve determination; ii. Based on the detection, melting curve, or absence of the (multiple) amplification products, differentially identify the presence of target nucleic acid in the sample, or determine that no target nucleic acid can be detected; as well as iii. Output real-time PCR results of the samples in the reaction vessel during or after the amplification, and output the melting results of the samples in the reaction vessel during or after the melting probe assay. e. A communication interface for communicating with an electronic device, the electronic device including a display screen with a user interface that displays one or more graphical elements, allowing the user to input a selection corresponding to at least one of a plurality of target assays, the at least one target assay being intended to determine the presence, absence or amount of at least one target nucleic acid in the biological sample.

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