Methods and systems for detecting pathogens

Abstract

To provide methods and systems for detecting pathogens. [Solution] A method, composition, and system for detecting the presence or absence of pathogens in a sample is disclosed. The method may include the steps of obtaining a sample from a subject and treating the sample with heat to inactivate any pathogens present in the sample. The method may further include the steps of treating the sample to concentrate any pathogens present in the sample. The method may also include isolating pathogen-specific nucleic acids from the heat-inactivated sample and detecting the presence or absence of the isolated pathogen-specific nucleic acids. In certain embodiments, SARS-CoV-2 can be detected using the method and / or system. The method can provide results in about 3 hours or less using real-time RT-PCR.

Description

Technical Field

[0001] Related Applications This application claims priority to U.S. Provisional Patent Application No. 63 / 004,143, filed Apr. 2, 2020, and U.S. Provisional Patent Application No. 63 / 058,172, filed Jul. 29, 2020. The disclosures of U.S. Provisional Patent Application No. 63 / 004,143 and No. 63 / 058,172 are hereby incorporated herein by reference in their entireties.

[0002] Sequence Listing This application includes a sequence listing submitted electronically in ASCII format, which is hereby incorporated herein by reference in its entirety. The name of the ASCII copy created on Mar. 30, 2021 is 057618-1235025_SL.txt, and the size is 3,388 bytes.

[0003] Field Methods, compositions, and systems related to testing for pathogens, including viral pathogens such as SARS-CoV-2 and variants thereof, are disclosed.

Background Art

[0004] Background SARS-CoV-2 is an enveloped, single-stranded RNA virus belonging to the genus Betacoronavirus in the family Coronaviridae. All coronaviruses share similarities in the composition and expression of their genomes, which encode 16 non-structural proteins and 4 structural proteins: spike (S), envelope (E), membrane (M), and nucleocapsid (N). Viruses in this family are zoonotic. They cause illnesses with symptoms ranging from mild colds to more severe conditions such as severe acute respiratory syndrome (SARS), Middle East respiratory syndrome (MERS), and coronavirus disease 2019 (COVID-19). Other coronaviruses known to infect humans include 229E, NL63, OC43, and HKU1. The latter is ubiquitous, and infection typically causes cold or flu-like symptoms (Su S, Wong G, Shi W, et al., Epidemiology, Genetic Recombination, and Pathogenesis of Coronaviruses, Trends Microbiol 2016;24(6):490-502; Zhu N, Zhang D, Wang W, et al., A Novel Coronavirus from Patients with Pneumonia in China, 2019. N Engl J Med 2020;382(8):727-733).

[0005] The SARS-CoV-2 virus can cause serious or life-threatening illnesses or symptoms, including severe respiratory illness, in humans infected with this virus. On February 11, 2020, the virus, initially tentatively named 2019-nCoV, was officially named Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2). Also on February 11, 2020, the disease caused by SARS-CoV-2 was officially named Coronavirus Disease 2019 (COVID-19). On February 4, 2020, the Secretary of Health and Human Services (HHS) determined that there was a public health emergency involving the virus causing COVID-19 with significant potential to affect national security or health and safety of U.S. citizens living abroad. Furthermore, new variants of SARS-CoV-2 have been detected. Therefore, there is a need for the development of methods and systems to detect pathogens such as SARS-CoV-2 and its variants. [Prior art documents] [Non-patent literature]

[0006] [Non-Patent Document 1] Su S, Wong G, Shi W, et al., Epidemiology, Genetic Recombination, and Pathogenesis of Coronaviruses, Trends Microbiol 2016;24(6):490-502 [Non-Patent Document 2] Zhu N,Zhang D,Wang W,et al.,A Novel Coronavirus from Patients with Pneumonia in China,2019.N Engl J Med 2020;382(8):727-733 [Overview of the project] [Means for solving the problem]

[0007] Abstract A system and method for detecting pathogens such as SARS-CoV-2 and its variants are disclosed. This method and system can be implemented in various ways.

[0008] In certain embodiments, the method may include a method for detecting the presence or absence of pathogens in a sample derived from a subject, comprising the steps of: obtaining a sample from a subject; processing the sample to inactivate any pathogens present in the sample; optionally processing the sample to concentrate any pathogens present in the sample; processing the thermally inactivated and optionally concentrated sample to isolate pathogen-specific nucleic acids from the sample; and detecting the presence or absence of the isolated pathogen-specific nucleic acids. In certain embodiments, the sample may be heated to inactivate pathogens. Additionally and / or alternatively, a protease may be added to the sample to inactivate pathogens.

[0009] In one embodiment, the pathogen is SARS-CoV-2. For example, in a particular embodiment, the method may include a method for detecting SARS-CoV-2 in a sample derived from a subject, comprising the steps of: obtaining a sample from a subject; isolating SARS-CoV-2 RNA from the sample; optionally treating the sample to inactivate the virus; generating copy DNA (cDNA) from the SARS-CoV-2 RNA; amplifying at least one target sequence of the SARS-CoV-2 cDNA; and detecting the amplified SARS-CoV-2 sequence. In a particular embodiment, the at least one target sequence of SARS-CoV-2 includes at least a portion of the nucleocapsid gene.

[0010] A system for carrying out the methods of this specification is also disclosed. For example, the system may comprise one or more stations for carrying out various steps of the method. In certain embodiments, the stations may comprise robotic stations for carrying out one or more steps. Furthermore, the system may comprise a computer program product tangibly embodied on a non-temporary machine-readable storage medium, which includes instructions configured to carry out the system or any part of the system, and / or instructions configured to carry out one or more steps of the method of any of the disclosed embodiments. In certain embodiments, for example, the following items are provided: (Item 1) A method for detecting SARS-CoV-2 in a sample derived from a target, A step of obtaining a sample from the aforementioned target; A step of isolating SARS-CoV-2 RNA from the aforementioned sample; A step of generating copy DNA (cDNA) from the aforementioned SARS-CoV-2 RNA; A step of amplifying at least one target sequence of the SARS-CoV-2 cDNA; and A method comprising the step of detecting the amplified SARS-CoV-2 sequence. (Item 2) The method according to item 1, wherein the sample is treated to inactivate the virus. (Item 3) The method according to item 2, wherein the sample is heated at 65°C for about 30 minutes in order to inactivate the virus. (Item 4) The method according to item 2, wherein the sample is treated with a protease to inactivate the virus. (Item 5) The method according to item 1, wherein the step of isolating SARS-CoV-2 RNA comprises, after the step of concentrating viral particles, eluting the SARS-CoV-2 RNA from the concentrated viral particles. (Item 6) The method according to item 5, wherein the elution of viral RNA from the concentrated viral particles is carried out at 95°C for at least 5 minutes. (Item 7) The method according to item 1, wherein the step of amplifying at least one target sequence of the SARS-CoV-2 cDNA includes quantitative PCR. (Item 8) The method according to item 1, wherein at least one target sequence of the SARS-CoV-2 cDNA comprises at least a portion of the SARS-CoV-2 nucleocapsid (N) gene. (Item 9) The method according to item 1, wherein the amplification step further comprises amplification of nucleic acid from a control gene that is not present in the virus but is present in the target. (Item 10) The method according to item 9, wherein the control gene is the human RNase P(RP) gene. (Item 11) The method according to item 1, wherein the sample includes a specimen originating from either the upper or lower respiratory tract. (Item 12) The method according to item 11, wherein the sample comprises at least one of the following: a nasopharyngeal swab, an oropharyngeal swab, sputum, a lower respiratory tract aspirate, a bronchoalveolar lavage fluid, a nasopharyngeal lavage or aspirate, or a nasal aspirate. (Item 13) The method according to item 1, wherein the step of amplifying at least one specific target sequence of the SARS-CoV-2 cDNA includes hybridizing a probe to the at least one specific target sequence, thereby causing the 5' nuclease activity of Taq polymerase to degrade the bound probe during the amplification extension step, separating the reporter dye on the probe from the quencher dye on the probe, and generating a fluorescent signal. (Item 14) The method according to item 13, wherein the reporter dye is FAM. (Item 15) The method according to item 13, wherein the quencher dye is BHQ1. (Item 16) The method according to item 1, wherein the step of amplifying at least one target sequence of SARS-CoV-2 comprises multiplex RT-PCR using primers and probes for SARS-CoV-2 N1, N2 and N3 sequences. (Item 17) The method according to item 1, wherein the step of amplifying at least one target sequence of SARS-CoV-2 comprises the use of at least one primer and / or probe having any one of the sequences of SEQ ID NOs: 1 to 9. (Item 18) A method for detecting the presence or absence of a pathogen in a sample derived from a subject, comprising: obtaining a sample from the subject; processing the sample to inactivate any pathogen present in the sample; optionally, processing the sample to concentrate any pathogen present in the sample; processing the inactivated and optionally concentrated sample to isolate pathogen-specific nucleic acid from the sample; and detecting the presence or absence of the isolated pathogen-specific nucleic acid. (Item 19) The method according to item 18, comprising: isolating RNA from the inactivated sample; generating copy DNA (cDNA) from the RNA isolated from the inactivated sample; amplifying at least one specific target sequence of the cDNA; and detecting the presence or absence of the amplified sequence. (Item 20) The method according to item 18, wherein the sample is heated to inactivate the pathogen. (Item 21) The method according to item 18, wherein the sample is treated with protease to inactivate the pathogen. (Item 22) The method according to item 18, wherein the pathogen is SARS-CoV-2. (Item 23) A system for detecting the presence or absence of pathogens in a sample derived from a target, comprising at least one station for inactivating the pathogens and a station for detecting the presence or absence of nucleic acids specific to the pathogens. (Item 24) A computer program product tangibly embodied in a non-temporary, machine-readable storage medium, containing instructions configured to detect the presence or absence of pathogen-specific nucleic acids. (Item 25) A kit containing reagents for detecting the presence or absence of SARS-CoV-2. (Item 26) The kit described in item 25 further comprises at least one primer and / or probe having one of the sequences of sequence numbers 1 to 9. (Item 27) A composition containing a reagent for detecting the presence or absence of SARS-CoV-2. (Item 28) The composition according to item 27, further comprising at least one primer and / or probe having one sequence of sequence numbers 1 to 9.

[0011] The disclosed methods and systems can be better understood by referring to the following non-limiting diagrams. [Brief explanation of the drawing]

[0012] [Figure 1] Figure 1 shows a method for detecting a pathogen according to one embodiment of the present disclosure. [Figure 2] Figure 2 shows an alternative method for detecting SAR-CoV-2 according to one embodiment of the present disclosure. [Figure 3] Figure 3 shows a system for detecting pathogens according to one embodiment of the present disclosure. [Figure 4] Figure 4 shows exemplary computing devices according to various embodiments of the present disclosure. [Modes for carrying out the invention]

[0013] Detailed explanation The following description provides only preferred exemplary embodiments and is not intended to limit the scope, applicability, or configuration of the Disclosure. Rather, the following description of preferred exemplary embodiments provides a possible description for carrying out various embodiments for those skilled in the art. It will be understood that various modifications can be made to the function and arrangement of the elements without departing from the spirit and scope set forth in the appended claims.

[0014] Specific details are given in the following description to provide a complete understanding of the embodiments. However, it will be understood that embodiments can be carried out without these specific details. For example, steps of a method, or parts of a system including circuits, systems, networks, processes, and other components, may be shown as components in block diagram form to avoid obscuring the embodiment with unnecessary details.

[0015] definition The Disclosure is described in more detail below. The Disclosure may be embodied in many different forms and should not be construed as being limited to the forms described herein. Rather, these forms are provided to satisfy the legal requirements to which the Disclosure applies. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which the Disclosure pertains. All patents, applications, published applications and other publications referenced herein are incorporated by reference in their entirety. If any definition in this section contradicts or is inconsistent with any definition in any patent, application, published application and other publication incorporated by reference herein, the definition in this section or any other part of this specification shall prevail.

[0016] Where introducing elements of this disclosure or its embodiments (singular or plural), the articles “a,” “an,” “the,” and “said” are intended to mean that there is one or more elements. The terms “comprising,” “including,” and “having” are intended to be comprehensive and mean that there may be additional elements other than those listed. The aspects and embodiments of the disclosure described herein are understood to include “consisting” and / or “consisting essentially of.”

[0017] The term "and / or," when used in a list of two or more items, means that any one of the listed items can be used alone or in combination with one or more of the listed items. For example, the expression "A and / or B" is intended to mean either A or B, or both, i.e., A alone, B alone, or a combination of A and B. The expression "A, B and / or C" is intended to mean A alone, B alone, C alone, a combination of A and B, a combination of A and C, a combination of B and C, or a combination of A, B, and C.

[0018] Various aspects of this disclosure may be presented in range form. It should be understood that range form descriptions are merely for convenience and conciseness and should not be interpreted as inflexible limitations on the scope of this disclosure. Therefore, range descriptions should be considered to specifically disclose all possible subranges and the individual numbers within those ranges. For example, a range description such as 1–6 should be considered to specifically disclose subranges such as 1–3, 1–4, 1–5, 2–4, 2–6, 3–6, and the individual numbers within those ranges, e.g., 1, 2, 3, 4, 5, and 6. This applies regardless of the width of the range.

[0019] As used herein, the terms “substantially,” “approximately,” and “about” are defined, as understood by those skilled in the art, as being largely but not necessarily the entirety of the specified thing (and including the entirety of the specified thing). In any disclosed embodiment, the terms “substantially,” “approximately,” or “about” may be replaced with “within [percentage]” of the specified thing, the percentages including 0.1, 1, 5, and 10%. As used herein, when an action is “based on” something, this means that the action is at least partially based on at least a part of something.

[0020] The terms “sample,” “patient sample,” “biological sample,” or “specimen” are used interchangeably herein. Samples may include upper and lower respiratory tract specimens. Such specimens (samples) may include nasopharyngeal or oropharyngeal swabs, sputum, lower respiratory tract aspirates, bronchoalveolar lavage fluid, and nasopharyngeal lavage / aspirates or nasal aspirates. Other non-limiting examples of samples include tissue samples (e.g., biopsies), blood or blood products (e.g., serum, plasma, etc.), cell-free DNA, urine, liquid biopsy samples, or combinations thereof. The term “blood” includes whole blood, blood products, or any fraction of blood such as serum, plasma, buffy coat, etc., as conventionally defined.

[0021] As used herein, the terms “subject” or “individual” refer to a human or any non-human animal. A subject or individual may be a patient, referring to a human being presented to a healthcare provider for the diagnosis or treatment of a disease, and in some cases, the disease may be any infectious disease caused by a pathogen. Also as used herein, the terms “individual,” “subject” or “patient” include all warm-blooded animals.

[0022] As used herein, “pathogen-specific nucleic acid” or “pathogen nucleic acid” refers to a nucleic acid molecule that is a sequence found in the pathogen genome but is not normally present in the subject. For example, “SARS-CoV-2-specific nucleic acid” or “SARS-CoV nucleic acid” is a sequence derived from the SARS-CoV-2 genome but is not normally found in the human genome (or a sample derived from a human subject).

[0023] As used herein, “SARS-CoV-2” or “SARS-CoV-2 virus” includes all genetic variants of the virus, including those that can cause the disease of COVID-19.

[0024] As used herein, the term “nucleic acid” refers to polynucleotides such as deoxyribonucleic acid (DNA) or ribonucleic acid (RNA). The term is used to include single-stranded nucleic acids, double-stranded nucleic acids, mRNA, and RNA and DNA made from nucleotides or nucleoside analogs.

[0025] As used herein, “detectable portion” is a chemical portion that enables quantitative measurement of a bound molecule. In certain embodiments, certain molecules used in accordance with and / or provided by the present invention (e.g., nucleic acid probes) comprise one or more detectable entities or portions, i.e., such molecules are “labeled” with such entities or portions. Any of the wide variety of detectable agents can be used in the practice of this disclosure. Suitable detectable agents include, but are not limited to, various ligands; radioactive nucleotides; fluorescent dyes; chemiluminescent agents (e.g., acridium esters, stabilized dioxetanes, etc.); bioluminescent agents; spectrally decomposable inorganic fluorescent semiconductor nanocrystals (i.e., quantum dots); microparticles; metal nanoparticles (e.g., gold, silver, copper, platinum, etc.); nanoclusters; paramagnetic metal ions; enzymes; colorimetric labels (e.g., dyes, gold colloids, etc.); biotin; dioxygenin; haptens; and proteins for which antiserum or monoclonal antibodies are available.

[0026] In certain embodiments, the detectable portion is a fluorescent dye. A large number of known fluorescent dyes with diverse chemical structures and physical properties are suitable for use in the implementation of this disclosure. The fluorescently detectable portion can be stimulated by a laser using light emitted by a detector. The detector may be a charge-coupled device (CCD) or a confocal microscope that records its intensity.

[0027] Suitable fluorescent dyes include fluorescein and fluorescein dyes (e.g., fluorescein isothiocyanine or FITC, naphthofluorescein, 4',5'-dichloro-2',7'-dimethoxyfluorescein, 6-carboxyfluorescein or FAM, etc.), hexachlorofluorescein (HEX), carbocyanine, merocyanine, styryl dyes, oxonol dyes, phycoerythrin, erythrosine, eosin, and rhodamine dyes (e.g., carboxytetramethylrhodamine or TAMRA, carboxyrhodamine). Rhodamine 6G, Carboxy-X-Rhodamine (ROX), Lisamin Rhodamine B, Rhodamine 6G, Rhodamine Green, Rhodamine Red, Tetramethylrhodamine (TMR), etc.), Coumarin and coumarin dyes (e.g., Methoxycoumarin, Dialkylaminocoumarin, Hydroxycoumarin, Aminomethylcoumarin (AMCA), etc.), Q-DOTS, Oregon Green dyes (e.g., Oregon Green 488, Oregon Green 500, Oregon Green 514, etc.), Texas Red, Texas Red-X, Spectrum RED, SPECTRUM GREEN, cyanine pigments (e.g., CY-3, CY-5, CY-3.5, CY5.5, etc.), ALEXA FLUOR pigment (e.g., Alexa Fluor 350, Alexa Fluor 488, Alexa Fluor 532, Alexa Fluor 546, Alexa Fluor 568, Alexa Examples of fluorescent labels include, but are not limited to, Fluor 594, Alexa Fluor 633, Alexa Fluor 660, Alexa Fluor 680, etc., BODIPY dyes (e.g., BODIPY FL, BODIPY R6G, BODIPY TMR, BODIPY TR, BODIPY 530 / 550, BODIPY 558 / 568, BODIPY 564 / 570, BODIPY 576 / 589, BODIPY 581 / 591, BODIPY 630 / 650, BODIPY 650 / 665, etc.), and IRDyes (e.g., IRD40, IRD 700, IRD 800, etc.). Preferred properties of fluorescent labels include high molar absorption coefficient, high fluorescence quantum yield, and photostability. In some embodiments, the labeled fluorophores exhibit absorption and emission wavelengths in the visible range (i.e., between 400 and 750 nm) rather than in the ultraviolet range of the spectrum (i.e., below 400 nm).

[0028] The detectable portion may contain more than one chemical entity, such as fluorescence resonance energy transfer (FRET). Resonance transfer results in an overall increase in emission intensity. To achieve resonance energy transfer, a first fluorescent molecule ("donor" phosphor) absorbs light and transfers it to a second fluorescent molecule ("acceptor" phosphor) through resonance of excited electrons. One approach is to ligate both the donor and acceptor dyes together and bind them to an oligoprimer. Examples of donor / acceptor pairs of dyes that can be used include fluorescein / tetramethylrhodamine, IAEDANS / fluorescein, EDANS / DABCYL, fluorescein / fluorescein, BODIPY FL / BODIPY FL, and fluorescein / QSY 7 dyes. Many of these dyes are commercially available, for example, from Molecular Probes Inc. (Eugene, Oreg.). Suitable donor fluorophores include 6-carboxyfluorescein (FAM), tetrachloro-6-carboxyfluorescein (TET), and 2'-chloro-7'-phenyl-1,4-dichloro-6-carboxyfluorescein (VIC).

[0029] Alternatively, a suitable fluorescent quencher molecule may be used. As used herein, fluorescent quenching refers to any process that reduces the fluorescence of a molecule, such as a black hole quencher commercially available from Biosearch Technologies. Such quenchers include, but are not limited to, BHQ0, BHQ1, BHQ3, and BHQ4. Various quencher dyes are suitable for use with certain fluorophores, including FAM, TET, JOE, HEX, Oregon Green®, TAMRA, ROX, cyanine-3, cyanine-3.5, cyanine-5, and cyanine-5.5 (e.g., CY-3, CY-5, CY-3.5, CY-5.5, etc.).

[0030] In certain embodiments, the detectable portion is an enzyme. Suitable enzymes include, but are not limited to, those used in enzyme-linked immunosorbent assays (ELISA), such as horseradish peroxidase, β-galactosidase, luciferase, and alkaline phosphatase. Other examples include β-glucuronidase, β-D-glucosidase, urease, and glucose oxidase. The enzyme may be linked to the molecule using a linker group such as carbodiimide, diisocyanate, or glutaraldehyde.

[0031] In certain embodiments, the detectable portion is a radioactive isotope. For example, the molecule may be isotope-labeled (i.e., it may contain one or more atoms replaced by atoms having atomic masses or mass numbers different from those commonly found in nature), or the isotope may be bound to the molecule. Non-restrictive examples of isotopes that can be incorporated into molecules include those of hydrogen, carbon, fluorine, phosphorus, copper, gallium, yttrium, technetium, indium, iodine, rhenium, thallium, bismuth, astatine, samarium, and lutetium (i.e., 3H, 13C, 14C, 18F, 19F, 32P, 35S, 64Cu, 67Cu, 67Ga, 90Y, 99mTc, 111In, 125I, 123I, 129I, 131I, 135I, 186Re, 187Re, 201T1, 212Bi, 213Bi, 21lAt, 153Sm, 177Lu).

[0032] Methods for detecting pathogens A system and method for detecting pathogens such as SARS-CoV-2 are disclosed. This method and system can be implemented in various ways.

[0033] In certain embodiments, the method may include a step of detecting the presence or absence of pathogens in a sample derived from a target, comprising the steps of: obtaining a sample from a target; processing the sample to inactivate any pathogens present in the sample; processing the sample to concentrate any pathogens present in the sample as needed; processing the thermally inactivated and optionally concentrated sample to isolate nucleic acids from the sample; and detecting the presence or absence of isolated pathogen-specific nucleic acids.

[0034] Various sample types can be used. In certain embodiments, the sample includes specimens originating from either the upper or lower respiratory tract. For example, the sample may be a nasopharyngeal or oropharyngeal swab, sputum, lower respiratory tract aspirate, bronchoalveolar lavage fluid, or nasopharyngeal lavage / aspirate or nasal aspirate. Alternatively, other types of samples may be used.

[0035] In certain embodiments, the detection step further includes amplification of a pathogen-specific sequence. For example, if the pathogen is a virus, the detection may include amplification of a virus-specific sequence. In certain embodiments where the pathogen is an RNA virus, the detection may include generating a virus-specific copy DNA (cDNA) sequence, followed by amplification, for example, by polymerase chain reaction (PCR) amplification of the virus-specific sequence. For example, the method may include the steps of isolating RNA from an inactivated sample, generating copy DNA (cDNA) from the RNA isolated from the inactivated sample, amplifying at least one specific target sequence of the cDNA, and detecting the presence or absence of the amplified sequence.

[0036] In certain embodiments, the detection step further includes amplification of a control gene that is present in the target but not in the pathogen. For example, the control gene may be another human gene, such as the human RNase P (RP) gene or a housekeeping gene involved in basic cell maintenance.

[0037] In some embodiments, the sample is heated to inactivate the pathogen. In alternative embodiments, the sample is heated to at least 60°C, or at least 65°C, or at least 70°C, or at least 75°C for a specified time. The sample may be heated for at least 10 minutes, or at least 20 minutes, or at least 30 minutes, or at least 40 minutes, or at least 50 minutes, or 1 hour, or longer. In one embodiment, the sample may be heated at 65°C for about 30 minutes. In certain embodiments, the sample is treated with a protease to inactivate the pathogen. In one embodiment, a protease, such as proteinase K, is also added to the sample before thermal inactivation. Other proteases may also be used.

[0038] In some embodiments, for the analysis of viral RNA, the step of isolating viral RNA includes nucleic acid extraction. Additionally and / or alternatively, the sample may be subjected to a method of first concentrating the pathogen. For example, to isolate viral particles, the sample may be subjected to viral concentration (e.g., purification) using a matrix designed to bind to viral particles (e.g., Nanotrap® Virus Capture Kit (Ceres Nanosciences, Inc.)). Using such a matrix, elution of viral RNA from the concentrated viral particles can be carried out at a temperature of about 90–99°C for at least 3 minutes. In one embodiment, elution may be carried out at 95°C for at least 5 minutes. Nucleic acids (e.g., RNA or DNA) can then be isolated from the sample.

[0039] Alternatively, other purification methods may be used. For example, a surfactant (e.g., lithium lauryl sulfate) with or without a protease (e.g., proteinase K), EDTA, and non-pathogenic DNA (e.g., salmon sperm) in an extraction buffer (e.g., HEPES buffer) can be used, incubated at approximately 60-65°C for approximately 1 hour, followed by extraction in phenol-chloroform-isoamyl alcohol and ethanol precipitation to isolate the nucleic acids. Alternatively, extraction may be carried out in the presence of guanidinium isothiocyanate or other chaotropic agents.

[0040] In one embodiment, the pathogen is SARS-CoV-2. Therefore, in a particular embodiment, the method may include a method for detecting SARS-CoV-2 in a sample derived from a subject, comprising the steps of: obtaining a sample from a subject; isolating SARS-CoV-2 RNA from the sample; generating copy DNA (cDNA) from the SARS-CoV-2 RNA; amplifying at least one target sequence of the SARS-CoV-2 cDNA; and detecting the amplified SARS-CoV-2 sequence. In one embodiment, at least one target sequence of the SARS-CoV-2 cDNA includes at least a portion of the SARS-CoV-2 nucleocapsid (N) gene.

[0041] This method may utilize quantitative reverse transcriptase (RT)PCR. For example, in certain embodiments, the step of amplifying at least one specific target sequence of a pathogen may include hybridizing a probe to at least one specific target sequence, thereby allowing the 5'→3' nuclease activity of Taq polymerase to degrade the bound probe during the amplification extension step, separating the reporter dye on the probe from the quencher dye on the probe during amplification, thereby generating a fluorescent signal. For the detection of SARS-CoV-2, the step of amplifying at least one target sequence of SARS-CoV-2 may include hybridizing a probe to at least one specific target sequence of SARS-CoV-2, thereby allowing the 5' nuclease activity of Taq polymerase to degrade the bound probe during the amplification extension step, separating the reporter dye on the probe from the quencher dye on the probe, thereby generating a fluorescent signal. Various reporter dyes and / or quenching dyes known in the art may be used. In certain embodiments, the reporter dye is FAM. Additionally and / or alternatively, the quencher dye may be BHQ1. For quantitative PCR, fluorescence intensity can then be monitored throughout the amplification process, for example, at each PCR cycle or at a selected time point.

[0042] A variety of pathogen-specific primers and probes can be used. For example, in the case of SARS-CoV-2, the step of amplifying at least one specific target sequence of SARS-CoV-2 includes primers and probes for COVID-19 N1, N2, and N3 targets, as well as multiplex RT-PCR using primers. In certain embodiments, the step of amplifying at least one specific target sequence of the SARS-CoV-2 nucleocapsid (N) gene present in cDNA includes the use of at least one primer and / or probe having any one of the sequences SEQ ID NOs. 1 to 9 disclosed herein.

[0043] In certain embodiments, the amplification step further includes amplification of a control gene that is not present in the virus but is present in the target gene. For example, the control gene may be the human RNase P (RP) gene or another gene such as a housekeeping gene. In certain embodiments, primers (SEQ ID NOs. 10 and 11) and the probe SEQ ID NO. 12 are used for the detection of the RP gene.

[0044] In certain embodiments, the assay is performed as a multiplex assay. For example, for the detection of SARS-CoV-2, the assay can be performed using three SARS-CoV-2 primers and probes as well as an RP primer and probe. Thus, in certain embodiments, the primers for the SARS-CoV-2 N1 gene are SEQ ID NOs: 1 and 2, and the internal probe is SEQ ID NO: 3. Also in certain embodiments, the primers for the SARS-CoV-2 N2 gene are SEQ ID NOs: 4 and 5, and the internal probe is SEQ ID NO: 6. Also in certain embodiments, the primers for the SARS-CoV-2 N3 gene are SEQ ID NOs: 7 and 8, and the internal probe is SEQ ID NO: 9. Also in certain embodiments, the primers for the RP gene are SEQ ID NOs: 10 and 11, and the internal probe is SEQ ID NO: 12. The TaqMan® probe may be labeled with Black Hole Quencher 1 (BHQ-1) (Biosearch Technologies, Inc., Novato, CA), which has its 5' end labeled with the reporter molecule 6-carboxyfluorescein (FAM) and its 3' end labeled with a quencher. These primer and probe variants can be used to detect SARS-CoV-2 mutants.

[0045] Fluorescence intensity can be monitored in each PCR cycle. The method may be automated. For example, in certain embodiments, fluorescence intensity during PCR amplification can be monitored using an Applied Biosystems QuantStudio 7 Flex (QS7) instrument with software version 1.3. Alternatively, other instruments and computer software for monitoring quantitative PCR may be used.

[0046] One embodiment of the method of the present disclosure (102) is shown in Figure 1. Thus, in one embodiment, the sample is obtained from the subject (104). In a particular embodiment, the sample may be a nasopharyngeal or oropharyngeal swab, sputum, lower respiratory tract aspirate, bronchoalveolar lavage fluid, or nasopharyngeal lavage / aspirate or nasal aspirate. Alternatively, other types of samples may be used.

[0047] Next, the sample can be treated with heat to inactivate pathogens present in the sample (106). In an alternative embodiment, the sample is heated to at least 60°C, or at least 65°C, or at least 70°C, or at least 75°C for a specified time. The sample may be heated for at least 10 minutes, or at least 20 minutes, or at least 30 minutes, or at least 40 minutes, or at least 50 minutes, or 1 hour, or longer. In one embodiment, the sample may be heated at 65°C for about 30 minutes. In one embodiment, a protease, such as proteinase K or another protease, can be added to inactivate the pathogens. In certain embodiments, the protease is also added to the sample before heat is applied for thermal inactivation.

[0048] Furthermore, if necessary, any pathogens present in the sample may be partially purified (e.g., concentrated) from the rest of the sample (108). For example, to isolate viral particles, the sample can be subjected to viral concentration (e.g., purification) using a matrix designed to bind to viral particles (e.g., Nanotrap® Virus Capture Kit, Ceres Nanosciences, Inc.). Alternatively, other purification methods may be used. Using such a matrix, elution of viral RNA from concentrated viral particles can be carried out at a temperature of 90–99°C for at least 3 minutes. In one embodiment, elution may be carried out at 95°C for at least 5 minutes. The nucleic acid (e.g., RNA or DNA) can then be isolated from the sample (110). At this point, the presence and / or amount of pathogen-specific nucleic acid can be determined (112). In certain embodiments where the pathogen nucleic acid is RNA, cDNA of at least a portion of the RNA may be produced.

[0049] For the detection of pathogen nucleic acids, pathogen-specific sequences can be amplified for subsequent detection. For example, in one embodiment, quantitative (i.e., real-time) PCR amplification can be used using primers and internal probes specific to the nucleic acid sequence in the pathogen. In one embodiment, the internal probe may be labeled with a reporter dye and a quencher dye so that the amplification allows the 5'→3' exonuclease activity of Taq polymerase to release the reporter dye, thereby allowing the amplification to be monitored. Any reporter dye and quencher dye in the art may be used. In a particular embodiment, the reporter dye is FAM and the quencher dye is BHQ1. Alternatively, other detection methods such as allele-type PCR amplification, digital PCR, or nucleic acid sequencing may be used. In a particular embodiment, the detection step further includes amplification of a control gene that is not present in the virus but is present in the target (112). For example, the control gene may be the human RNase P (RP) gene, or another gene such as a housekeeping gene.

[0050] At this point, the results may be reported to the subject, their healthcare provider, or other healthcare professional (114).

[0051] The method may be automated. For example, in one embodiment, cDNA is amplified using an Applied Biosystems QuantStudio 7 Flex (QS7) instrument with software version 1.3. Alternatively, other amplification systems may be used.

[0052] Figure 2 shows one embodiment (200) of the method of the present disclosure for detecting SARS-CoV-2. Thus, in one embodiment, the sample is obtained from the subject (202). In a particular embodiment, the sample may be a nasopharyngeal or oropharyngeal swab, sputum, lower respiratory tract aspirate, bronchoalveolar lavage fluid, or nasopharyngeal lavage / aspirate or nasal aspirate. Alternatively, other types of samples may be used.

[0053] The sample can be treated with heat as needed to inactivate the SARS-CoV-2 virus present in the sample (204). In an alternative embodiment, the sample is heated to at least 60°C, or at least 65°C, or at least 70°C, or at least 75°C for a specified time. The sample may be heated for at least 10 minutes, or at least 20 minutes, or at least 30 minutes, or at least 40 minutes, or at least 50 minutes, or 1 hour, or longer. In one embodiment, the sample may be heated at 65°C for about 30 minutes. Thus, in one embodiment, the sample can be heated to inactivate the SARS-CoV-2 virus. In one embodiment, a protease, such as proteinase K or another protease, is also added to inactivate the virus. The protease may be added before heating so that it functions during the thermal inactivation of the virus.

[0054] Furthermore, if necessary, the SARS-CoV-2 virus present in the sample may be partially purified (e.g., concentrated) from the rest of the sample (206). For example, the sample can be subjected to viral concentration (e.g., purification) using a matrix designed to bind to viral particles (e.g., Nanotrap® Virus Capture Kit, Ceres Nanosciences, Inc.) to isolate the viral particles. Alternatively, other purification methods may be used. Using such a matrix, elution of viral RNA from the concentrated viral particles can be carried out at a temperature in the range of 90–99°C for at least 3 minutes. In one embodiment, elution may be carried out at 95°C for at least 5 minutes or under similar conditions.

[0055] At this point, SARS-CoV-2 RNA can be isolated from the sample (208), and this can be used to generate copy DNA (cDNA) (210). The cDNA can be used to determine the presence and / or amount of the SARS-CoV-2 RNA sequence in the sample. For example, in one embodiment, quantitative PCR amplification is used (212). Quantitative PCR can be performed by amplifying at least one specific target sequence of the SARS-CoV-2 nucleocapsid (N) gene present in the cDNA and detecting the amplified SARS-CoV-2 nucleocapsid (N) gene sequence. In one embodiment, an internal probe that can bind to the cDNA or amplification product may be labeled with a reporter dye and a quencher dye so that the amplification can be monitored, allowing the 5' exonuclease activity of Taq polymerase to release the reporter dye. Any reporter dye and quencher dye in the art may be used. In a particular embodiment, the reporter dye is FAM and the quencher dye is BHQ1. In certain embodiments, the primers and probes (SEQ ID NOs. 1-9) shown in Table 2 are used for the detection of SARS-CoV-2. In certain embodiments, the amplification step further includes the amplification of a control gene that is not present in the virus but is present in the target. For example, the control gene may be the human RNase P (RP) gene or another gene. In certain embodiments, the primers (SEQ ID NOs. 10-11) and the probe of SEQ ID NO. 12 are used for the detection of the RP gene. The results may be reported to the subject, their healthcare provider, or other healthcare professional (214).

[0056] The method and system can be optimized to yield results in less than one day. For example, in the case of the analysis of SARS-CoV-2 disclosed herein, the method may take less than 10 hours, or less than 8 hours, or less than 6 hours, or less than 4 hours, or less than 3 hours, or less than 2 hours, or less than 1 hour. Also, as described above, in one embodiment, the sample may be subjected to viral concentration and / or thermal inactivation and / or protease treatment before extraction of viral nucleic acid. In one embodiment, thermal inactivation allows for improved sample processing and / or protects laboratory personnel. This can improve throughput and allow processing of multiple samples (e.g., 400 samples) in about 40 minutes or less. For example, in certain embodiments, the disclosed method may be performed robotically. Robotic processing may allow for the analysis of a large number of samples with shorter turnaround times than methods performed entirely manually. In robotic embodiments, the sample can be robotically extracted from a sample collection device (e.g., tubes, vials, sample carriers, etc.) for further analysis by any of the methods disclosed herein. In further robotic embodiments, the extracted sample can be placed in a reaction vessel (e.g., a tube, vial, etc.) for a PCR reaction by any of the disclosed methods. Additionally and / or alternatively, the PCR reaction may be performed by the robot.

[0057] Compositions and kits Compositions and / or kits for carrying out any of the disclosed methods or for performing any of the disclosed systems are also disclosed herein. In one embodiment, the composition and / or kit comprises a reagent for detecting the presence or absence of pathogens in a sample derived from a subject. The composition and / or kit may further comprise reagents or components for obtaining a sample from the subject, e.g., nasal swabs, buffers, preservatives, etc. The composition and / or kit may further comprise reagents or components for thermally treating the sample to inactivate any pathogens present in the sample. The composition and / or kit may further comprise reagents or components for treating the sample with a protease. The composition and / or kit may further comprise reagents or components for partially purifying pathogens, as will be discussed in detail herein. The reagents and / or components may be individually packaged. The composition and / or kit may also further comprise instructions for use.

[0058] The composition and / or kit may further include reagents or components for detecting the presence or absence of isolated pathogen-specific nucleic acids. For example, in some embodiments, the composition and / or kit may further include reagents or components for generating copy DNA (cDNA) from pathogen-specific (and / or control) RNA.

[0059] Additionally and / or alternatively, the composition and / or kit may further comprise reagents or components for quantitative PCR amplification using primers and internal probes specific to nucleic acid sequences in the pathogen. In certain embodiments, the internal probe may be labeled with a reporter dye and a quencher dye so that amplification can monitor the release of the reporter dye of the 5'→3' exonuclease activity of Taq polymerase. Any reporter dye and quencher dye in the art may be used. In certain embodiments, the reporter dye is FAM and the quencher dye is BHQ1.

[0060] In some embodiments, for the detection of SARS-CoV-2, the composition and / or kit may further include reagents or components for amplifying at least one specific target sequence of the SARS-CoV-2 nucleocapsid (N) gene present in cDNA and for detecting the amplified SARS-CoV-2 nucleocapsid (N) gene sequence.

[0061] The compositions and / or kits of this disclosure may include a variety of primers and probes specific to SARS-CoV-2. In certain embodiments, the compositions and / or kits may include primers and / or probes for SARS-CoV-2 N1, N2, and N3 targets. In certain embodiments, the step of amplifying at least one specific target sequence of the SARS-CoV-2 nucleocapsid (N) gene present in cDNA includes the use of at least one primer and / or probe having one of the sequences of SEQ ID NOs. 1 to 9 disclosed herein. Also in certain embodiments, the compositions and / or kits may include reagents (e.g., primers and probes) for amplifying a control gene that is not present in the virus but is present in the target. For example, the control gene may be the human RNase P (RP) gene or another gene. For detection of the RP sequence, the reagents (e.g., primers and probes) for amplifying the RP gene may include the primers of SEQ ID NOs. 10 and 11 and the probe of SEQ ID NO. 12. In certain embodiments, the primers and / or probes are labeled with detectable regions, such as the detectable regions disclosed herein.

[0062] The composition and / or kit can be used to reverse transcribe RNA into cDNA for subsequent amplification using quantitative PCR. For the analysis of SARS-CoV-2, RT-PCR may involve a multiplex reaction using primers and probes for SARS-CoV-2 and / or primers and probes for an internal (i.e., non-SARS-CoV-2) control, such as the human RP gene. Alternatively, other combinations of primers and probes may be used for other pathogens. In certain embodiments of the disclosed composition and / or kit, the assay is performed as a multiplex assay using three sets of SARS-CoV-2 primers and probes for the nucleocapsid gene and one set of primers and probes for the RP gene. Thus, in certain embodiments, the primers for the SARS-CoV-2 N1 gene are SEQ ID NOs: 1 and 2, and the internal probe is SEQ ID NO: 3. Also, in certain embodiments, the primers for the SARS-CoV-2 N2 gene are SEQ ID NOs: 4 and 5, and the internal probe is SEQ ID NO: 6. In certain embodiments, the primers for the SARS-CoV-2 N3 gene are SEQ ID NOs. 7 and 8, and the internal probe is SEQ ID NO. 9. Also in certain embodiments, the primers for the RP gene are SEQ ID NOs. 10 and 11, and the internal probe is SEQ ID NO. 12. The TaqMan® probe may be labeled with Black Hole Quencher 1 (BHQ-1) (Biosearch Technologies, Inc., Novato, CA), which has its 5' end labeled with the reporter molecule 6-carboxyfluorescein (FAM) and its 3' end labeled with a quencher. Alternatively, other dyes may be used. Alternatively, the primers may be labeled with any of the detectable portions disclosed herein for detection of the amplification product without requiring an internal probe. Furthermore, variants of these primers and probes can be used to detect SARS-CoV-2 mutants.

[0063] In certain embodiments, the composition and / or kit may include at least one of the following controls:

[0064] Internal control – RNase P (RP) control in clinical samples. RP primer and probe sets may be included in each run to test for human RP to control sample quality and demonstrate that nucleic acids were generated by the extraction process. Alternatively, other internal controls may be used.

[0065] A positive template control is provided, containing an in vitro transcription template (e.g., SARS-CoV-2) RNA with the genomic region targeted by this method. The positive control may be used to monitor failures of the rRT-PCR reagent and reaction conditions. Alternatively, a different positive template control may be used.

[0066] Negative Extraction Control (NEC) – In one embodiment, this may be a previously characterized negative patient sample. The NEC can be used as an extraction control and positive control for internal (e.g., RP) primer and probe sets.

[0067] Nuclease-free molecular-grade water can be used to monitor template-free (negative) control-nonspecific amplification, cross-contamination during experimental setup, and nucleic acid contamination of reagents.

[0068] system A system for carrying out the method described herein is also disclosed. For example, the system may comprise one or more stations for carrying out various steps of the method. In certain embodiments, the stations may comprise a robotic station for carrying out one or more steps of the method.

[0069] Figure 3 shows one embodiment of a system (300) for pathogen detection. Thus, the system may include a station (302) for acquiring and / or receiving a sample from a target.

[0070] For example, in many cases, samples can be collected from the subject (e.g., by a medical professional, caregiver, or the subject themselves) at a location away from the test area and sent to the test area. The sample may be a nasopharyngeal or oropharyngeal swab, sputum, lower respiratory tract aspirate, bronchoalveolar lavage fluid, or nasopharyngeal lavage / aspirate or nasal aspirate. Alternatively, other types of samples may be used.

[0071] In certain embodiments, the system may have a station (304) for processing the sample to inactivate pathogens present in the sample. In certain embodiments, the sample is heated to inactivate the pathogens. In alternative embodiments, the sample is heated to at least 60°C, or at least 65°C, or at least 70°C, or at least 75°C for a specified time. The sample may be heated for at least 10 minutes, or at least 20 minutes, or at least 30 minutes, or at least 40 minutes, or at least 50 minutes, or 1 hour, or longer. In one embodiment, the sample may be heated at 65°C for about 30 minutes. In certain embodiments, the system may have a station for adding a protease, such as proteinase K or another protease. This station (not shown in Figure 3) may be before, after, or part of the station for thermal inactivation.

[0072] Furthermore, the system may optionally include a station (306) for partially purifying (e.g., concentrating) any pathogens present in the sample. For example, to isolate viral particles, the sample can be subjected to viral concentration (e.g., purification) using a matrix designed to bind to viral particles (e.g., Nanotrap® Virus Capture Kit, Ceres Nanosciences, Inc.). Alternatively, other purification methods may be used. Using such a matrix, elution of viral RNA from the concentrated viral particles can be carried out at a temperature of 90–99°C for at least 3 minutes. In one embodiment, elution may be carried out at 95°C for about 5 minutes.

[0073] The system may also have a station (308) for isolating nucleic acids (e.g., RNA or DNA) from a sample. If the nucleic acid is RNA, the system may have a station (310) for generating cDNA from the RNA. The system may also have a station (312) for determining the presence and / or amount of pathogen-specific nucleic acids in the sample. For example, in one embodiment, quantitative PCR amplification using primers and internal probes specific to the nucleic acid sequence in the pathogen may be used, as disclosed herein. The system may also include a station (314) for reporting the results to the subject, their healthcare provider, or other healthcare professional.

[0074] This disclosure takes into consideration that some of the stations, such as those illustrated, may be combined as a single station. For example, but not limited to, stations for RNA isolation, cDNA preparation, and quantitative PCR may be combined as a single station. Alternatively, stations for thermal inactivation and partial purification of pathogens may be combined. Also, as shown in Figure 3, any of the stations may be automated, robotically controlled, and / or at least partially controlled by a computer (400) and / or programmable software. Thus, the system may include a computer program product tangibly embodied in a non-temporary machine-readable storage medium, which includes instructions configured to perform the system or any part of the system, and / or instructions configured to perform one or more steps of any of the methods of any of the disclosed embodiments. In some embodiments, a system is provided which includes one or more data processors and a non-temporary computer-readable storage medium containing instructions that, when executed on the one or more data processors, cause the one or more data processors to perform some or all of the methods or processes disclosed herein.

[0075] For example, a system is disclosed comprising one or more data processors and a non-temporary computer-readable storage medium containing instructions that cause one or more data processors to perform at least one of the following actions when performed on one or more data processors: taking a sample from a subject; processing the sample to inactivate any pathogens present in the sample; processing the sample to concentrate any pathogens present in the sample, if necessary; processing the inactivated and optionally concentrated sample to isolate pathogen-specific nucleic acids from the sample; and detecting the presence or absence of the isolated pathogen-specific nucleic acids.

[0076] Also disclosed are computer program products tangibly embodied in a non-temporary machine-readable storage medium, which include instructions configured to run a system and / or instructions configured to perform one or more steps of any of the methods of the disclosed embodiments. For example, in certain embodiments, the computer program product tangibly embodied in a non-temporary machine-readable storage medium includes instructions configured to cause one or more data processors to perform at least one of the following steps: taking a sample from a subject; processing the sample to inactivate any pathogens present in the sample; processing the sample to concentrate any pathogens present in the sample, if necessary; processing the inactivated and optionally concentrated sample to isolate pathogen-specific nucleic acids from the sample; and detecting the presence or absence of the isolated pathogen-specific nucleic acids.

[0077] The system and computer products can perform any of the methods disclosed herein. One or more embodiments described herein can be carried out using program modules, engines, or components. Program modules, engines, or components may include programs, subroutines, parts of programs, or software or hardware components capable of performing one or more of the described tasks or functions. When used herein, a module or component may reside on a hardware component independently of other modules or components. Alternatively, a module or component may be a shared element or process of another module, program, or machine.

[0078] Figure 4 shows a block diagram of an analytical system (400) used for the detection and / or quantification of pathogens. As shown in Figure 4, various subsystems of the analytical instrument system according to various embodiments can be implemented using modules, engines, or components (e.g., programs, code, or instructions) that can be executed by one or more processors. The modules, engines, or components may be stored on non-temporary computer media. If necessary, one or more of the modules, engines, or components can be loaded into system memory (e.g., RAM) and executed by one or more processors of the analytical instrument system. An example shown in Figure 4 illustrates modules, engines, or components for carrying out the method of the present disclosure.

[0079] Accordingly, Figure 4 shows an exemplary computing device (400) suitable for use in the systems and methods of the present disclosure. The exemplary computing device (400) comprises a processor (405) communicating with memory (410) and other components of the computing device (400) using one or more communication buses (415). The processor (405) is configured to execute processor executable instructions stored in memory (410) to perform one or more methods for detecting pathogen levels or to operate one or more stations according to different examples such as those in Figures 1-3 or disclosed elsewhere in this specification. In this example, memory (410) may store processor executable instructions (425) that can analyze (420) RT-PCR results for SARS-CoV-2 or other pathogens, as discussed herein.

[0080] The computing device 400 in this example may also include one or more user input devices (430), such as a keyboard, mouse, touchscreen, or microphone, to accept user input. The computing device (400) may also include a display (435) to provide the user with visual output, such as a user interface. The computing device (400) may also include a communication interface (440). In some examples, the communication interface (440) can enable communication over one or more networks, including a local area network ("LAN"); a wide area network ("WAN") such as the Internet; a metropolitan area network ("MAN"); point-to-point or peer-to-peer connections, etc. Communication with other devices can be achieved using any suitable network protocol. For example, one suitable network protocol could include the Internet Protocol ("IP"), Transmission Control Protocol ("TCP"), User Datagram Protocol ("UDP"), or a combination thereof, such as TCP / IP or UDP / IP.

[0081] Detection of SARS-CoV-2 One embodiment of the disclosed SARS-CoV-2 RT-PCR test includes a real-time reverse transcription polymerase chain reaction (rRT-PCR) test for the qualitative detection of nucleic acids from SARS-CoV-2 in upper and lower respiratory tract specimens (e.g., nasopharyngeal or oropharyngeal swabs, sputum, lower respiratory tract aspirates, bronchoalveolar lavage fluid, and nasopharyngeal lavage / aspirates or nasal aspirates) collected from individuals suspected of being infected with SARS-CoV-2.

[0082] In one embodiment, the results are presented for the identification of SARS-CoV-2 RNA. SARS-CoV-2 RNA is generally detectable in respiratory specimens during the acute phase of infection. A positive result indicates the presence of SARS-CoV-2 RNA. In some embodiments, the patient's infectious status can be determined using clinical correlation with the patient's medical history and other diagnostic information. In one embodiment, a positive result does not rule out co-infection with bacterial infection or other viruses. The detected drug may not be the clear cause of the disease. Also, in one embodiment, a negative result does not rule out SARS-CoV-2 infection and should not be used as the sole basis for determining patient management. A negative result should be combined with clinical findings, the patient's medical history, and epidemiological information.

[0083] As disclosed herein, in some embodiments, the method utilizes a step of thermal inactivation of the pathogen. In one embodiment, after thermal inactivation of the virus, SARS-CoV-2 nucleic acid is isolated, extracted, and purified from upper and lower respiratory tract specimens (e.g., nasopharyngeal or oropharyngeal swabs, sputum, lower respiratory tract aspirates, bronchoalveolar lavage fluid, and nasopharyngeal lavage / aspirates or nasal aspirates). In some embodiments, the samples are subjected to a virus concentration method and / or thermal extraction of viral nucleic acid. The purified nucleic acid can then be reverse transcribed to cDNA, followed by PCR amplification and detection by one-step (multiplex) RT-PCR, as disclosed in detail herein.

[0084] In certain embodiments, the method utilizes at least one of the following controls.

[0085] Internal control – RNase P (RP) control in clinical samples. RP primer and probe sets are included in each run to test for human RP, which controls sample quality and demonstrates that nucleic acids were generated by the extraction process. Alternatively, other internal controls may be used.

[0086] A positive template control is provided, containing an in vitro transcription template (e.g., SARS-CoV-2) RNA with the genomic region targeted by this method. The positive control may be used to monitor failures of the rRT-PCR reagent and reaction conditions.

[0087] Negative Extraction Control (NEC) – In one embodiment, this may be a previously characterized negative patient sample. It is used as an extraction control and positive control for the RP primer and probe set.

[0088] Nuclease-free molecular-grade water used for template-free (negative) control-nonspecific amplification, cross-contamination during experimental setup, and nucleic acid contamination of reagents. [Examples]

[0089] Example 1 The samples were divided equally (e.g., into 96-well plates), and the virus was then inactivated by heating at 65°C for 30 minutes in the presence of proteinase K. The COVID-19 RT-PCR test can be performed with Roche MagNA Pure-96 (MP96) using an Applied Biosystems QuantStudio7 Flex (QS7) instrument equipped with MagNA Pure 96 DNA and Viral NA Small Volume Kit and software version 1.3. Primers and probes are those recommended by the Centers for Disease Control and Prevention (CDC). Tables 1 and 2 provide the primers, probes, and reagents that can be used. The assay was performed as a multiplex assay using all three SARS-CoV-2 primers and probes, as well as RP primers and probes. The TaqMan® probe was labeled at its 5' end with the reporter molecule 6-carboxyfluorescein (FAM) and at its 3' end with the quencher Black Hole Quencher 1 (BHQ-1) (Biosearch Technologies, Inc., Novato, CA). Y = pyrimidine. In one embodiment, the oligonucleotide sequence may be modified for the detection of SARS-CoV-2 mutants. [Table 1] [Table 2]

[0090] Control used in COVID-19 RT-PCR 1) A negative (untemplated) control is used to eliminate the possibility of sample contamination during assay execution and is used in all assay plates. This control is molecular-grade nuclease-free water. 2) A positive template (COVID-19_N_P) control is used to verify that the assay run is performed as intended and is used in all assay plates starting with the addition of the master mix at a concentration of 50 copies / uL. The positive control is prepared from in vitro transcribed and purified viral RNA targets containing one copy each of N1, N2, and N3. The positive template control does not contain the RNase P target and is obtained as “undetermined” for its marker. 3) An internal (Hs_RPP30) control targeting RNase P is used to verify that nucleic acids are present in all samples and is used for all processed samples. This also serves as an extraction control to ensure that samples obtained as negative contain nucleic acids for testing. 4) The negative extract (NEC) control is a previously characterized negative patient sample. This serves both as a negative extract control to monitor any cross-contamination that may occur during the extraction process, and as an extraction control to validate the extraction reagent and successful RNA extraction.

[0091] Interpretation of results Before interpreting patient results, examine all test controls. If the controls are ineffective, patient results are ininterpretable or generally uninterpretable.

[0092] 1) COVID-19 RT-PCR test controls - positive, negative, and internal: Negative (no template control) - Negative for all detected targets (Ct not detected). Positive (COVID-19_N_P) - Positive for all detected targets (Ct<40), Internal Extract (Hs_RPP30) - Negative for SARS-CoV-2 target (Ct not detected), positive for RNase P(RP) target (Ct<40). Negative extract (NEC)-SARS-CoV-2 target was negative (Ct not detected), while the RNase P (RP) target was positive (Ct < 40). If control is not performed as described above, the execution is considered invalid, and all samples are repeated from the extraction process.

[0093] 2) Review and interpretation of patient sample results: RP - All clinical samples should yield a positive result for the RP target with a Ct of less than 40. Samples that fail to show detection of RP and all three SARS-CoV-2 targets within this range should be repeated from the extraction process. If a sample detects any of the SARS-CoV-2 targets, the lack of amplification of the RP target may be valid (Table 3). [Table 3]

[0094] Performance evaluation 1) Analysis sensitivity: Limit of Detection (LoD): The LoD study established the lowest concentration (genomic copy (cp) / μL) of SARS-CoV-2 that could be detected by COVID-19 RT-PCR testing at a rate of at least 95%. The preliminary LoD was established by testing 10-fold dilutions of SARS-CoV-2 synthetic RNA. The preliminary LoD was confirmed by testing 2-fold dilutions of 20 replicates (50 cp / μL, 25 cp / μL, 12.5 cp / μL, 6.25 cp / μL, 3.125 cp / μL, 1.25 cp / μL). 2-fold dilution samples were prepared by adding quantified live SARS-CoV-2 to negative respiratory clinical matrix (NP swabs and BALs). The study results showed a COVID-19 RT-PCR LoD of 6.25 cp / μL (19 / 20 positive).

[0095] 2) Analysis specificity: Cross-reactivity of COVID-19 RT-PCR tests was evaluated using in silico analysis and by testing whole organisms or purified nucleic acids from a panel of organisms listed in the table below. Empirical testing showed that all targets were negative for all microorganisms tested, with the exception of SARS coronavirus, which is expected to react with the N3 target of the COVID-19 RT-PCR test (the target for universal detection of SARS-like viruses) (Table 4; ND = not detected). [Table 4]

[0096] BLAST analysis did not show homology between the organisms listed in Table 5 and the primers and probes used in the COVID-19 RT-PCR test. [Table 5]

[0097] 3) Clinical evaluation: A devised clinical trial was conducted to evaluate the performance of the COVID-19 RT-PCR test. A total of 100 individual clinical respiratory samples, 50 NP (nasopharyngeal) swabs, and 50 BAL (bronchial alveolar lavage) samples were used in this study. 100 negative and 80 artificially positive samples were tested. Negative samples consisted of 50 NP swabs and 50 BAL samples. Positive samples consisted of 40 NP swabs and 40 BAL samples spiked with quantified live SARS-CoV-2. Ten samples were spiked with 8×, 4×, 2×, and 1× LOD, respectively. In one devised BAL sample prepared with LoD, the N3 target was not determined. The agreement between the positive and negative percentages between the COVID-19 RT-PCR test and the predicted results from NP swabs and BAL samples is shown in Tables 6 and 7. [Table 6] [Table 7]

[0098] Furthermore, five positive patient samples and five negative patient samples were sent to the North Carolina Department of Health (NCDOH) and tested using CDC assays under EUA. All results were consistent (Table 8). [Table 8]

[0099] Example 2 In certain embodiments, the method may be carried out by concentrating viral particles and subsequently extracting viral RNA from the viral particles. Once the RNA has been extracted, amplification can be performed using the primers and method detailed in Example 1.

[0100] Briefly, the sample (nasal swab) is received and a portion is dispensed into individual wells of a plate (e.g., a 96-well microtiter plate). After inactivating the viral proteins by treating with proteinase K and heating at 65°C for 30 minutes (as in Example 1), the sample is subjected to viral enrichment (e.g., purification) using a matrix designed to bind to viral particles (e.g., Nanotrap® Virus Capture Kit, Ceres Nanosciences, Inc.). Briefly, the heat-inactivated viral particles are mixed with virus capture beads (e.g., Nanotrap®) as recommended by the manufacturer. The beads can then be magnetically concentrated, the medium removed, and the beads washed with phosphate-buffered saline (PBS). After magnetic concentration, elution buffer is added, and the viral particles attached to the beads are incubated in the elution buffer for the required time (e.g., 5 minutes) at 95°C. The beads are then magnetically concentrated, viral RNA removed, and transferred to another plate for RT-PCR as described in Example 1. The process can be automated using a Hamilton robot. Using thermal extraction of the virus, processing of 400 samples can be completed in 40 minutes, compared to 4 hours using the purification method described in Example 1.

[0101] Example 3 - Embodiment Various non-limiting embodiments are provided below.

[0102] A1. A method for detecting SARS-CoV-2 in a sample derived from a target, The process of obtaining a sample from the target; The process of isolating SARS-CoV-2 RNA from a sample; The process of generating copy DNA (cDNA) from SARS-CoV-2 RNA; A step of amplifying at least one target sequence of SARS-CoV-2 cDNA; and A method comprising the step of detecting an amplified SARS-CoV-2 sequence.

[0103] A2. A method according to any one of the embodiments of the method described above or below, wherein the sample is treated to inactivate the virus.

[0104] A3. A method according to any one of the embodiments of the method described above or below, wherein the sample is heated to inactivate the virus.

[0105] A4. A method according to any one embodiment of the method described above or below, wherein the sample is heated to at least 60°C, or at least 65°C, or at least 70°C, or at least 75°C for a specified time.

[0106] A4.1 The method according to any one of the embodiments of the method described above or below, wherein the sample is heated for at least 10 minutes, or at least 20 minutes, or at least 30 minutes, or at least 40 minutes, or at least 50 minutes, or 1 hour, or longer.

[0107] A4.2 The method according to any one of the embodiments of the method described above or below, wherein the sample is heated at 65°C for about 30 minutes to inactivate the virus.

[0108] A5. The method according to any one of the embodiments of the method described above or below, wherein the sample is treated with a protease to inactivate the virus.

[0109] A6. A method according to any one of the embodiments of the method described above or below, wherein the sample is treated with proteinase K.

[0110] A7. A method according to any one embodiment of the method described above or below, wherein the step of isolating SARS-CoV-2 RNA includes, after the step of concentrating viral particles, elution of SARS-CoV-2 RNA from the concentrated viral particles.

[0111] A8. The method according to any one of the embodiments of the method described above or below, wherein viral RNA is eluted from concentrated viral particles at 95°C for at least 5 minutes.

[0112] A9. A method according to any one of the embodiments of the method described above or below, wherein the step of amplifying at least one target sequence of SARS-CoV-2 includes quantitative RT-PCR.

[0113] A10. The method according to any one embodiment of the method described above or below, wherein at least one target sequence of SARS-CoV-2 comprises at least a portion of the nucleocapsid (N) gene of SARS-CoV-2.

[0114] A11. At least one target sequence of SARS-CoV-2 A method according to any one of the embodiments of the method described above or below, comprising the sequences N1, N2, and N3.

[0115] A12. A method according to any one embodiment of the method described above or below, wherein the step of amplifying at least one target sequence of SARS-CoV-2 comprises multiplex RT-PCR using primers and probes for SARS-CoV-2 N1, N2, and N3 sequences.

[0116] A13. The method according to any one embodiment of the preceding or subsequent embodiments of the method, comprising the step of amplifying at least one specific target sequence of SARS-CoV-2 cDNA, wherein the probe is hybridized to at least one specific target sequence, thereby causing the 5' nuclease activity of Taq polymerase to degrade the bound probe during the amplification extension step, separating the reporter dye on the probe from the quencher dye on the probe, and generating a fluorescent signal.

[0117] A14. The method according to any one of the embodiments of the method described above or below, wherein the reporter dye is FAM.

[0118] A15. The method according to any one of the embodiments of the method described above or below, wherein the quencher dye is BHQ1.

[0119] A16. A method according to any one of the embodiments of the preceding or following methods, wherein the amplification step further comprises amplification of a nucleic acid sequence from a control gene that is not present in the virus but is present in the target.

[0120] A17. The method according to any one of the embodiments described above or below, wherein the control gene is the human RNase P(RP) gene.

[0121] A18. The method according to any one embodiment of the method described above or below, wherein the step of amplifying at least one target sequence of SARS-CoV-2 includes the use of at least one primer and / or probe having one of sequence numbers 1 to 9.

[0122] A19. The method according to any one of the embodiments of the method described above or below, wherein the step of amplifying a nucleic acid sequence from the RP gene includes the use of at least one primer and / or probe having one of the sequences of sequence numbers 10 to 12.

[0123] A20. A method according to any one embodiment of the method described above or below, wherein the sample includes a specimen originating from either the upper or lower respiratory tract.

[0124] A21. The method according to any one embodiment of the method described above or below, wherein the sample comprises at least one of the following: a nasopharyngeal swab, an oropharyngeal swab, sputum, a lower respiratory tract aspirate, a bronchoalveolar lavage fluid, a nasopharyngeal lavage or aspirate, or a nasal aspirate.

[0125] B1. A method for detecting the presence or absence of pathogens in a sample derived from a target, The process of obtaining a sample from the target; A process of treating a sample to inactivate any pathogens present in the sample; A step of processing the sample to concentrate any pathogens present in the sample, if necessary; A step of processing the sample, which has been inactivated and, if necessary, concentrated, in order to isolate pathogen-specific nucleic acids from the sample; and A method comprising the step of detecting the presence or absence of isolated pathogen-specific nucleic acids.

[0126] B2. Steps to isolate RNA from the inactivated sample; The process of generating copy DNA (cDNA) from RNA isolated from an inactivated sample; A step of amplifying at least one specific target sequence of cDNA; and A method according to any one embodiment of the method described above or below, further comprising the step of detecting the presence or absence of an amplified sequence.

[0127] B3. A method according to any one embodiment of the method described above or below, wherein the pathogen is SARS-CoV-2.

[0128] B4. A method according to any one of the embodiments of the method described above or below, wherein the sample is heated to inactivate the pathogen.

[0129] B5. A method according to any one embodiment of the method described above or below, wherein the sample is heated to at least 60°C, or at least 65°C, or at least 70°C, or at least 75°C for a specified time.

[0130] B5.1 The method according to any one of the embodiments of the method described above or below, wherein the sample is heated for at least 10 minutes, or at least 20 minutes, or at least 30 minutes, or at least 40 minutes, or at least 50 minutes, or 1 hour, or longer.

[0131] B5.2 The method according to any one of the embodiments of the method described above or below, wherein the sample is heated at 65°C for about 30 minutes to inactivate the pathogen.

[0132] B6. The method according to any one of the embodiments of the method described above or below, wherein the sample is treated with a protease to inactivate the pathogen.

[0133] B7. A method according to any one of the embodiments of the method described above or below, wherein the sample is treated with proteinase K.

[0134] B8. A method according to any one of the embodiments of the method described above or below, wherein the step of isolating pathogen nucleic acids includes elution of pathogen nucleic acids after the step of concentrating inactivated pathogens.

[0135] B9. The method according to any one of the embodiments of the method described above or below, wherein the elution of pathogenic nucleic acids from the concentrated pathogen is carried out at 95°C for at least 5 minutes.

[0136] B10. A method according to any one of the embodiments described above or below, wherein the step of amplifying at least one target sequence of pathogen nucleic acid includes quantitative PCR.

[0137] B11. The method according to any one of the embodiments of the method described above or below, wherein at least one target sequence of SARS-CoV-2 comprises at least a portion of the nucleocapsid (N) gene of SARS-CoV-2.

[0138] B12. At least one target sequence of SARS-CoV-2 A method according to any one of the embodiments of the method described above or below, comprising the sequences N1, N2, and N3.

[0139] B13. A method according to any one of the embodiments of the method described above or below, wherein the step of amplifying at least one target sequence of a pathogen comprises multiplex RT-PCR using primers and probes for one or more target sequences of a pathogen.

[0140] B14. The method according to any one embodiment of the method described above or below, wherein the step of amplifying at least one target sequence of a pathogen comprises hybridizing a probe to at least one target sequence of a pathogen, thereby causing the 5' nuclease activity of Taq polymerase to degrade the bound probe during the amplification extension step, separating the reporter dye on the probe from the quencher dye on the probe, and generating a fluorescent signal.

[0141] B15. The method according to any one of the embodiments of the method described above or below, wherein the reporter dye is FAM.

[0142] B16. The method according to any one of the embodiments of the method described above or below, wherein the quencher dye is BHQ1.

[0143] B17. A method according to any one of the embodiments of the preceding or following methods, wherein the amplification step further comprises amplification of a nucleic acid sequence from a control gene that is not present in the virus but is present in the target.

[0144] B18. The method according to any one of the embodiments described above or below, wherein the control gene is the human RNase P(RP) gene.

[0145] B19. The method according to any one of the embodiments of the method described above or below, wherein the step of amplifying at least one target sequence of SARS-CoV-2 includes the use of at least one primer and / or probe having one of the sequences of sequence numbers 1 to 9.

[0146] The method according to any one of the embodiments of the method described above or below, wherein the step of amplifying a nucleic acid sequence from the B20.RP gene includes the use of at least one primer and / or probe having one of the sequences of sequence numbers 10 to 12.

[0147] B21. The method according to any one embodiment of the method described above or below, wherein the sample includes a specimen originating from either the upper or lower respiratory tract.

[0148] B22. The method according to any one embodiment of the method described above or below, wherein the sample comprises at least one of the following: a nasopharyngeal swab, an oropharyngeal swab, sputum, a lower respiratory tract aspirate, a bronchoalveolar lavage fluid, a nasopharyngeal lavage or aspirate, or a nasal aspirate.

[0149] C1. A system for carrying out the steps or any of the steps of the embodiments described above, or for using any of the compositions and / or kits of the embodiments of the kits or compositions described below.

[0150] C2. A system for detecting the presence or absence of pathogens in a sample derived from a target, comprising at least one station for inactivating pathogens and a station for detecting the presence or absence of nucleic acids specific to the pathogen.

[0151] C3. A system, either of the embodiments described above or below, comprising a station for receiving or acquiring a sample from a subject.

[0152] C4. A system, according to any of the embodiments of the system described above or below, comprising a station for purifying or partially purifying pathogens from a sample derived from the target.

[0153] C5. A system, either of the embodiments described above or below, comprising a station for isolating nucleic acids from pathogens.

[0154] C6. A system, either of the above or below embodiments of the system, comprising a station for reporting results.

[0155] C7. A system according to any of the embodiments of the system described above or below, wherein the pathogen is SARS-CoV-2.

[0156] C8. A system according to any of the embodiments of the system described above or below, wherein the sample is heated to inactivate the pathogen.

[0157] C9. The system according to any one of the embodiments of the system described above or below, wherein the sample is heated to at least 60°C, or at least 65°C, or at least 70°C, or at least 75°C for a specified time.

[0158] C9.1 A system according to any of the embodiments of the system described above or below, wherein the sample is heated for at least 10 minutes, or at least 20 minutes, or at least 30 minutes, or at least 40 minutes, or at least 50 minutes, or 1 hour, or longer.

[0159] C9.2 A system according to any of the embodiments of the system described above or below, wherein the sample is heated at 65°C for approximately 30 minutes to inactivate the pathogen.

[0160] C10. A system according to any of the embodiments of the system described above or below, wherein the sample is treated with a protease to inactivate the pathogen.

[0161] C11. A system according to any of the embodiments of the system described above or below, wherein the sample is treated with proteinase K.

[0162] C12. A system according to any of the embodiments of the system described above or below, wherein the step of isolating pathogen nucleic acids includes elution of pathogen nucleic acids after the step of concentrating inactivated pathogens.

[0163] C13. A system according to any of the embodiments of the system described above or below, wherein the elution of pathogenic nucleic acids from concentrated pathogens is performed at 95°C for at least 5 minutes.

[0164] C14. A system according to any of the embodiments of the system described above or below, wherein the step of amplifying at least one target sequence of pathogen nucleic acid includes quantitative PCR.

[0165] A system according to any of the embodiments of the system described above or below, wherein at least one target sequence of SARS-CoV-2 comprises at least a portion of the SARS-CoV-2 nucleocapsid (N) gene.

[0166] C16. At least one target sequence of SARS-CoV-2 A system according to any of the embodiments of the system described above or below, comprising the sequences N1, N2, and N3.

[0167] C17. A system according to any of the embodiments of the system described above or below, wherein the step of amplifying at least one target sequence of a pathogen includes multiplex RT-PCR using primers and probes for one or more target sequences of a pathogen.

[0168] C18. A system according to any embodiment of the system described above or below, wherein the step of amplifying at least one target sequence of a pathogen comprises hybridizing a probe to at least one target sequence of a pathogen, thereby causing the 5' nuclease activity of Taq polymerase to degrade the bound probe during the amplification extension step, separating the reporter dye on the probe from the quencher dye on the probe, and generating a fluorescent signal.

[0169] C19. The system according to any of the embodiments of the system described above or below, wherein the reporter dye is FAM.

[0170] C20. The system according to any of the embodiments of the system described above or below, wherein the quencher dye is BHQ1.

[0171] C21. The system according to any of the embodiments of the system described above or below, wherein the amplification step further comprises the amplification of a nucleic acid sequence from a control gene that is not present in the virus but is present in the target.

[0172] C22. The system described in either of the embodiments of the system described above or below, wherein the control gene is the human RNase P(RP) gene.

[0173] The system according to any of the embodiments of the system described above or below, wherein the step of amplifying at least one target sequence of C23.SARS-CoV-2 includes the use of at least one primer and / or probe having one of sequence numbers 1 to 9.

[0174] The system according to any of the embodiments of the system described above or below, wherein the step of amplifying the nucleic acid sequence from the C24.RP gene includes the use of at least one primer and / or probe having one of the sequences of sequence numbers 10 to 12.

[0175] C25. A system according to any of the embodiments of the system described above or below, wherein the sample includes a specimen originating from either the upper or lower respiratory tract.

[0176] C26. A system according to any embodiment of the system described above or below, wherein the sample includes at least one of the following: a nasopharyngeal swab, an oropharyngeal swab, sputum, lower respiratory tract aspirate, bronchoalveolar lavage fluid, nasopharyngeal lavage fluid or aspirate, or nasal aspirate.

[0177] C27. A system according to any of the embodiments of the system described above or below, wherein at least one station is automated and / or controlled by a computer.

[0178] D1. A computer program product tangibly embodied in a non-temporary machine-readable storage medium, comprising instructions configured to perform any step of the present method or to execute any of the stations of the system of the embodiments described above.

[0179] D2. A computer program product tangibly embodied in a non-temporary, machine-readable storage medium, which includes instructions configured to detect the presence or absence of nucleic acids specific to a pathogen.

[0180] A computer program product tangibly embodied in a non-temporary machine-readable storage medium, comprising instructions configured to detect the presence or absence of D3.SARS-CoV-2 nucleic acid, as described in either the preceding or subsequent embodiments.

[0181] E1. A composition comprising a reagent for carrying out any of the methods of the embodiments described above.

[0182] A composition according to any embodiment of the composition described above or below, comprising a reagent for detecting the presence or absence of E2.SARS-CoV-2.

[0183] E3. A composition according to any embodiment of the composition described above or below, further comprising at least one primer and / or probe having one sequence of any of sequence numbers 1 to 9.

[0184] E4. A composition according to any embodiment of the composition described above or below, further comprising at least one primer and / or probe having one sequence of any of sequence numbers 10 to 12.

[0185] E5. A composition according to any of the embodiments of the composition described above or below, comprising a reagent for obtaining a sample from a subject.

[0186] E6. A composition according to any embodiment of the composition described above or below, comprising a protease for inactivating a pathogen.

[0187] E7. A composition according to any of the embodiments of the compositions described above or below, comprising a reagent for purifying or partially purifying a pathogen from other components in a sample.

[0188] E8. A composition according to any of the embodiments of the compositions described above or below, comprising a reagent for detecting a pathogen.

[0189] E9. A composition according to any of the embodiments of the compositions described above or below, comprising a reagent for generating cDNA from RNA.

[0190] E10. A composition according to any of the embodiments of the compositions described above or below, comprising a reagent for quantitative PCR.

[0191] E11. A composition according to any embodiment of the composition described above or below, wherein a probe for quantitative PCR is labeled with a reporter dye and a quencher dye.

[0192] E12. A composition according to any embodiment of the composition described above or below, wherein a probe for quantitative PCR is labeled with FAM and / or BHQ1.

[0193] E13. A composition according to any of the embodiments of the composition described above or below, including instructions for using any of the reagents.

[0194] E14. A composition according to any of the embodiments of the compositions described above or below, wherein the reagent or its components are packaged in individual containers.

[0195] F1. A kit containing reagents for carrying out any of the methods of the embodiments described above.

[0196] A kit according to any embodiment of the kit described above or below, comprising a reagent for detecting the presence or absence of F2.SARS-CoV-2.

[0197] A kit according to any embodiment of the kit described above or below, further comprising at least one primer and / or probe having one of the sequences of sequence numbers 1 to 9.

[0198] F4. A kit of any embodiment of the kit described above or below, further comprising at least one primer and / or probe having one of the sequences of sequence numbers 10 to 12.

[0199] F5. A kit according to one of the embodiments of the kit described above or below, comprising reagents or components for obtaining a sample from a subject.

[0200] F6. A kit according to any embodiment of the kit described above or below, comprising a protease for inactivating a pathogen.

[0201] F7. A kit according to any embodiment of the kit described above or below, comprising reagents for purifying or partially purifying pathogens from other components in the sample.

[0202] F8. A kit according to any of the embodiments of the kit described above or below, comprising reagents for detecting pathogens.

[0203] A kit according to one of the embodiments of the kit described above or below, comprising reagents for generating cDNA from F9 RNA.

[0204] F10. A kit according to any of the embodiments of the kit described above or below, comprising reagents for quantitative PCR.

[0205] F11. A kit according to any embodiment of the kit described above or below, wherein the probe for quantitative PCR is labeled with a reporter dye and a quencher dye.

[0206] F12. A kit according to any embodiment of the kit described above or below, wherein the probe for quantitative PCR is labeled with FAM and / or BHQ1.

[0207] F13. A kit of any of the embodiments of the kit described above or below, including instructions for using any of the reagents.

[0208] F14. A kit according to any of the embodiments of the kit described above or below, wherein the reagent or its components are packaged in individual containers.

[0209] Further consideration The above description provides specific details to offer a complete understanding of the embodiments. However, it is understood that embodiments can be carried out without these specific details. For example, circuits can be shown in block diagrams to avoid obscuring the embodiments with unnecessary details. In other examples, well-known circuits, processes, algorithms, structures, and techniques can be shown without unnecessary details to avoid obscuring the embodiments.

[0210] The implementation of the technologies, blocks, processes, and means described above can be carried out in a variety of ways. For example, these technologies, blocks, processes, and means can be implemented in hardware, software, or a combination thereof. In the case of hardware implementation, the processing unit can be implemented in one or more application-specific integrated circuits (ASICs), digital signal processors (DSPs), digital signal processing devices (DSPDs), programmable logic devices (PLDs), field-programmable gate arrays (FPGAs), processors, controllers, microcontrollers, microprocessors, other electronic units designed to perform the functions described above, and / or a combination thereof.

[0211] Furthermore, it should be noted that embodiments can be described as processes shown as flowcharts, flow diagrams, data flow diagrams, structural diagrams, or block diagrams. While flowcharts can describe operations as sequential processes, many operations can be performed in parallel or simultaneously. Moreover, the order of operations can be rearranged. A process terminates when its operations are completed, but it may have additional steps not shown in the diagram. A process can correspond to a method, function, procedure, subroutine, subprogram, etc. If a process corresponds to a function, its termination corresponds to the function's return to the calling function or main function.

[0212] Furthermore, embodiments can be implemented by hardware, software, scripting languages, firmware, middleware, microcode, hardware description languages, and / or any combination thereof. When implemented by software, firmware, middleware, scripting languages, and / or microcode, program code or code segments for performing the required tasks can be stored in a machine-readable medium such as a storage medium. Code segments or machine-executable instructions can represent procedures, functions, subprograms, programs, routines, subroutines, modules, software packages, scripts, classes, or any combination of instructions, data structures, and / or program statements. Code segments can be coupled to other code segments or hardware circuits by passing and / or receiving information, data, arguments, parameters, and / or memory contents. Information, arguments, parameters, data, etc., can be passed, transferred, or transmitted via any suitable means, including memory sharing, message passing, ticket passing, network transmission, etc.

[0213] In the case of firmware and / or software implementations, the methodology may be implemented in modules (e.g., procedures, functions, etc.) that perform the functions described herein. When implementing the methodology described herein, any machine-readable medium that tangibly embodies the instructions may be used. For example, software code may be stored in memory. Memory may be implemented within or outside the processor. As used herein, the term “memory” means any type of long-term, short-term, volatile, non-volatile, or other storage medium, and is not limited to any particular type of memory or number of memories, or the type of medium in which the memory is stored.

[0214] Furthermore, as disclosed herein, the terms “storage medium,” “storage,” or “memory” may refer to one or more memories for storing data, including read-only memory (ROM), random access memory (RAM), magnetic RAM, core memory, magnetic disk storage medium, optical storage medium, flash memory devices, and / or other machine-readable media for storing information. The term “machine-readable media” includes, but is not limited to, portable or fixed storage devices, optical storage devices, wireless channels, and / or various other storage media capable of storing instructions (one or more) and / or data to be stored or carried.

[0215] While the principles of this disclosure are described above in relation to specific apparatuses and methods, it should be clearly understood that this description is provided for illustrative purposes only and not as a limitation on the scope of this disclosure.

Claims

[Claim 1] The invention described in the specification.

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