Compositions and methods for detecting severe acute respiratory syndrome coronavirus 2 (SARS-COV-2), influenza A, and influenza B

Abstract

Disclosed are methods for rapidly detecting the presence or absence of SARS-CoV-2, influenza A, and influenza B in biological or non-biological samples. The methods may include performing amplification, hybridization, and detection steps. Additionally, primers and probes targeting SARS-CoV-2, influenza A, and influenza B, as well as kits designed for detecting SARS-CoV-2, influenza A, and influenza B, are provided.

Description

[Technical Field]

[0001] FIELD OF THE INVENTION The present disclosure relates to the field of viral diagnostics, and more particularly to the detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) or SARS-CoV-2, influenza A virus (influenza A), and influenza B virus (influenza B). [Background technology]

[0002] Background of the Invention Viruses in the Coronaviridae family have single-stranded, positive-sense RNA genomes ranging in length from 26 to 32 kilobases. Coronaviruses have been identified in several avian hosts and in a variety of mammals, including camels, bats, palm civets, mice, dogs, and cats. Novel mammalian coronaviruses are now being identified regularly. For example, the HKU2-related coronavirus, which originated in bats, was responsible for a fatal acute diarrheal syndrome in pigs in 2018.

[0003] Among the several coronaviruses that are pathogenic to humans, most are associated with mild clinical symptoms, with two notable exceptions: severe acute respiratory syndrome (SARS) coronavirus (SARS-CoV), a novel betacoronavirus that emerged in Guangdong Province, southern China, in November 2002 and resulted in over 8,000 human infections and 774 deaths in 37 countries between 2002 and 2003; and Middle East respiratory syndrome (MERS) coronavirus (MERS-CoV), which was first detected in Arabia in 2012 and has been responsible for 2,494 laboratory-confirmed cases of infection and 858 deaths since September 2012, including 38 deaths after a single introduction into South Korea.

[0004] In late December 2019, several patients with viral pneumonia were epidemiologically linked to a market in Wuhan, Hubei Province, China, where many non-aquatic animals, such as birds and rabbits, were also sold prior to the outbreak. A novel human-infectious coronavirus, initially designated 2019 novel coronavirus (2019-nCoV), was identified using next-generation sequencing. This novel coronavirus is classified in the Coronaviridae family, the Betacoronavirus genus, and the Sarbecovirus subgenus. It is described in "Genomic characterization and epidemiology of 2019 novel coronavirus: implications for virus origins and receptor binding" by Lu, R. et al., Lancet, 2020, Vol. 395, pp. 565-574. The International Committee on Taxonomy of Viruses (ICTV) has officially named this virus severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). As of March 1, 2020, China has reported nearly 80,000 confirmed cases and over 2,800 deaths, and SARS-CoV-2 has been detected in at least 60 locations internationally, including the U.S. Therefore, there is a need in the art for rapid, reliable, specific, and sensitive methods for detecting SARS-CoV-2.

[0005] Influenza, or the flu, is a highly contagious viral respiratory disease of the Orthomyxoviridae family. Flu infections can occur at any time, but are usually characterized by seasonal outbreaks during the winter months in each hemisphere. Symptoms vary greatly in severity from patient to patient but typically include one or more of the following: cough, fever, runny or stuffy nose, sore throat, body aches, and fatigue. While anyone can be infected with the flu, it is particularly dangerous for the elderly and young children, as well as those with weakened immune systems and certain pre-existing conditions.

[0006] There are four known types of influenza viruses, designated influenza viruses A through D. Humans can be infected with influenza viruses A, B, and C, but no cases of influenza D infection in humans have been reported. The most common type infecting humans is influenza A, followed by influenza B. Influenza A is further divided into serotypes based on variations in two proteins found on the outer surface of the virus particle: hemagglutinin (H) and neuraminidase (N). Hemagglutinin variants H1 through H3 and neuraminidase variants N1 and N2 form the most common serotypes occurring during seasonal epidemics. Over the course of several years, influenza A epidemics have had devastating effects worldwide, resulting in influenza pandemics. For example, the 1918 Spanish flu pandemic is estimated to have killed 17 to 50 million people. The 1957 Asian flu pandemic and the 1968 Hong Kong flu pandemic each killed more than one million people. The influenza A H1N1 serotype was responsible for the 1918 Spanish flu, while the H2N2 and H3N2 serotypes were the causative agents of the Asian flu and Hong Kong flu pandemics, respectively.

[0007] Influenza B and influenza C can infect humans but are much less dangerous. Because influenza B has a single serotype, it is easier to establish and maintain herd immunity against this virus. Nevertheless, serious infections of children and adolescents, and even localized epidemics, can occur with this virus. Influenza C can infect humans but is even less dangerous than influenza B, with patients showing only mild symptoms.

[0008] Therefore, beyond the need for rapid, reliable, specific, and sensitive methods for detecting SARS-CoV-2 itself, rapid and accurate diagnosis and differentiation of SARS-CoV-2 and influenza infections is also important in individuals with suspected respiratory infections. The seasonal spectrum of SARS-CoV-2 and influenza overlaps, and the clinical symptoms of the two diseases can be similar, ranging from an asymptomatic or mild "flu-like" illness (e.g., fever, cough, shortness of breath, or myalgia) in the majority of individuals to more severe and life-threatening illness. However, the two virus types differ in that SARS-CoV-2 patients can spread infection while prodromal, while influenza patients develop symptoms more rapidly and do not shed virus while prodromal. Consequently, rapid and accurate detection and differentiation of both SARS-CoV-2 and influenza can help inform time-critical medical decision-making, facilitate infection control efforts, promote efficient resource delivery, optimize the use of targeted therapies and antimicrobials, and reduce ancillary testing or procedures. Therefore, there is a need in the art for rapid, reliable, specific and sensitive methods for detecting and distinguishing between SARS-CoV-2, influenza A and influenza B. Summary of the Invention

[0009] Summary of the Invention The present disclosure provides methods for rapid detection of the presence or absence of SARS-CoV-2 in biological or non-biological samples, e.g., multiplexed detection of SARS-CoV-2 by qualitative or quantitative real-time reverse transcription polymerase chain reaction (RT-PCR) in a single test tube or a single well. The present disclosure also provides methods for rapid and simultaneous detection of the presence or absence of SARS-CoV-2, influenza A, and influenza B in biological or non-biological samples, e.g., multiplexed detection of SARS-CoV-2, influenza A, and influenza B by qualitative or quantitative real-time reverse transcription polymerase chain reaction (RT-PCR) in a single test tube or a single well. Embodiments include methods for detecting SARS-CoV-2, influenza A, and influenza B, comprising performing a reverse transcription step and at least one cycling step, which may include an amplification step and a hybridization step. Additionally, embodiments include primers, probes, and kits designed for detecting SARS-CoV-2 or for multiplexed detection of SARS-CoV-2, influenza A, and influenza B in a single tube or a single well. Detection methods are designed to target various regions of each target genome. For example, the methods are designed to target regions of the SARS-CoV-2 genome encoding the structural envelope (E) region and / or the nonstructural open reading frame (ORF1a / b gene, which encodes the ORF1a polyprotein and ORF1a / b polyprotein). The methods can also be designed to target other regions of the SARS-CoV-2 genome, such as the S gene (encoding the spike protein involved in binding to cellular receptors), ORF3ab, the E gene (encoding the envelope protein), and the M gene (encoding the membrane protein), alone or in combination. Additionally, there is a 265-base noncoding region at the 5' end of the SARS-CoV-2 genome and a 229-base noncoding region at the 3' end, which may also be targeted.

[0010] For influenza A and B, the method can be designed to target any gene or non-coding region within the eight segments that make up their genomes. For example, for influenza A, the method can be designed to target the influenza A segment 7 matrix protein 2 (M2) and matrix protein 1 (M1) sequences. For influenza B, the method can be designed to target the influenza B segment 8 nuclear export protein (NEP) and nonstructural protein 1 (NS1) sequences.

[0011] The method for simultaneous detection of SARS-CoV-2, influenza A, and influenza B by RT-PCR in a single tube or well may also include simultaneous detection by RT-PCR in a single tube or well of additional viruses that can cause respiratory disease in humans, particularly in the upper respiratory tract. Examples of these viruses include, but are not limited to: coronaviruses (229E, NL63, OC43, HKU1), respiratory syncytial virus, human metapneumovirus, adenoviruses (B, E, U, C), enteroviruses, rhinoviruses, and human parainfluenza viruses (1, 2, 3, 4).

[0012] In one embodiment, there is provided a method for detecting SARS-CoV-2 in a sample, the method comprising: performing an amplification step comprising contacting the sample with a primer set to produce an amplification product if SARS-CoV-2 is present in the sample; performing a hybridization step comprising contacting the amplification product with one or more detectable probes; and detecting the presence or absence of the amplification product, wherein the presence of the amplification product indicates the presence of SARS-CoV-2 in the sample and the absence of the amplification product indicates the absence of SARS-CoV-2 in the sample; wherein the primer set comprises or consists of an oligonucleotide sequence selected from the group consisting of SEQ ID NOs: 1-20, 27-31 and 40-41, or a complement thereof; and the one or more detectable probes comprise or consist of an oligonucleotide sequence selected from the group consisting of SEQ ID NOs: 21-26, 32, and 42-43, or a complement thereof.

[0013] In one embodiment, there is provided a multiplex method for detecting SARS-CoV-2 in a sample, comprising: performing an amplification step comprising contacting the sample with a first primer set to produce an amplification product if SARS-CoV-2 is present in the sample, and contacting the sample with a second primer set to produce an amplification product if SARS-CoV-2 is present in the sample; performing a hybridization step comprising contacting the amplification product with at least one detectable probe that hybridizes to the amplification product produced by the first primer pair and at least one second detectable probe that hybridizes to the amplification product produced by the second primer pair; and detecting the presence or absence of the amplification product(s), wherein the presence of the amplification product(s) indicates the presence of SARS-CoV-2 in the sample and the absence of the amplification product indicates the absence of SARS-CoV-2 in the sample; the first primer set comprises or consists of a forward primer oligonucleotide sequence selected from the group consisting of SEQ ID NOS: 4 to 31 and a reverse primer oligonucleotide sequence selected from the group consisting of SEQ ID NOS: 7 to 14; the second primer set comprises or consists of a forward primer oligonucleotide sequence selected from the group consisting of SEQ ID NOS: 4 to 6, and 40 and a reverse primer oligonucleotide sequence selected from the group consisting of SEQ ID NOS: 15 to 20, and 41; at least one detectable probe that hybridizes to the amplification product produced by the first primer pair comprises or consists of an oligonucleotide sequence selected from the group consisting of SEQ ID NOS: 21 to 23, and 42, or a complement thereof; and at least one detectable probe that hybridizes to the amplification product produced by the second primer pair comprises or consists of an oligonucleotide sequence selected from the group consisting of SEQ ID NOS: 24 to 26, 32, and 43, or a complement thereof.

[0014] In some embodiments, a first primer pair specifically hybridizes to and amplifies a SARS-CoV-2 target nucleic acid, and a second primer pair hybridizes to and amplifies a SARS-CoV-2 target nucleic acid. In some embodiments, a second primer pair hybridizes to and amplifies a SARS-CoV-2 target nucleic acid and other coronavirus target nucleic acids from the subgenus Sarbecovirus. In some embodiments, at least one detectable probe that hybridizes to an amplification product produced by the first primer pair specifically hybridizes to a SARS-CoV-2 target nucleic acid. In some embodiments, at least one detectable probe that hybridizes to an amplification product produced by the second primer pair hybridizes to a SARS-CoV-2 target nucleic acid. In some embodiments, at least one detectable probe that hybridizes to an amplification product produced by the second primer pair hybridizes to a SARS-CoV-2 target nucleic acid and other coronavirus target nucleic acids from the subgenus Sarbecovirus.

[0015] In one embodiment, the primer set for amplification of a SARS-CoV-2 target comprises a first primer comprising or consisting of a first oligonucleotide sequence selected from the group consisting of SEQ ID NOs: 1-6, 27-31, and 40, or its complement, and a second primer comprising or consisting of a second oligonucleotide sequence selected from the group consisting of SEQ ID NOs: 7-20, and 41, or its complement, and one or more detectable probes for detection of the amplification product comprise or consist of an oligonucleotide sequence selected from the group consisting of SEQ ID NOs: 21-26, 32, 42, and 43, or its complement.

[0016] In one embodiment, the primer set for specific amplification of a SARS-CoV-2 target comprises a first primer comprising or consisting of a first oligonucleotide sequence selected from the group consisting of SEQ ID NOs: 1-3, and 27-31, and a second primer comprising or consisting of a second oligonucleotide sequence selected from the group consisting of SEQ ID NOs: 7-14, and one or more detectable probes for detection of the amplification product comprise or consist of an oligonucleotide sequence selected from the group consisting of SEQ ID NOs: 21-23, and 42, or a complement thereof.

[0017] In one embodiment, the primer set for specific amplification of a SARS-CoV-2 target comprises a first primer comprising or consisting of a first oligonucleotide sequence of SEQ ID NO: 40 and a second primer comprising or consisting of a second oligonucleotide sequence of SEQ ID NO: 41, and the one or more detectable probes for detection of the amplification product comprise or consist of the oligonucleotide sequence of SEQ ID NO: 43 or its complement.

[0018] In one embodiment, a primer set for amplification of a SARS-CoV-2 target comprises a first primer comprising or consisting of a first oligonucleotide sequence selected from the group consisting of SEQ ID NOs: 4-6 and a second primer comprising or consisting of a second oligonucleotide sequence selected from the group consisting of SEQ ID NOs: 15-20, and one or more detectable probes for detection of the amplification product comprise or consist of an oligonucleotide sequence selected from the group consisting of SEQ ID NOs: 24-26, and 32, or a complement thereof. In some embodiments, the primer set is suitable for amplification of SARS-CoV-2 target nucleic acids and other coronavirus target nucleic acids from the subgenus Sarbecovirus.

[0019] In another embodiment, a primer set for amplification of a SARS-CoV-2 target comprises a first primer comprising or consisting of the oligonucleotide sequence of SEQ ID NO: 1, a second primer comprising or consisting of the oligonucleotide sequence of SEQ ID NO: 7, and a detectable probe comprising or consisting of the oligonucleotide sequence of SEQ ID NO: 21. In another embodiment, the first primer comprises or consists of an oligonucleotide sequence selected from the group consisting of SEQ ID NOs: 27 and 30, the second primer comprises or consists of the oligonucleotide sequence of SEQ ID NO: 7, and the detectable probe comprises or consists of the oligonucleotide sequence of SEQ ID NO: 21. In another embodiment, a primer set for amplification of a SARS-CoV-2 target comprises a first primer comprising or consisting of the oligonucleotide sequence of SEQ ID NO: 1, a second primer comprising or consisting of the oligonucleotide sequence of SEQ ID NO: 7, 8, or 9, and a detectable probe comprising or consisting of the oligonucleotide sequence of SEQ ID NO: 21. In another embodiment, a primer set for amplification of a SARS-CoV-2 target comprises a first primer comprising or consisting of the oligonucleotide sequence of SEQ ID NO: 2, a second primer comprising or consisting of the oligonucleotide sequence of SEQ ID NO: 9, 10, or 11, and a detectable probe comprising or consisting of the oligonucleotide sequence of SEQ ID NO: 22.

[0020] In another embodiment, the first primer comprises or consists of the oligonucleotide sequence of SEQ ID NO:5, the second primer comprises or consists of the oligonucleotide sequence of SEQ ID NO:15, and the detectable probe comprises or consists of an oligonucleotide sequence selected from the group consisting of SEQ ID NOs:25 and 32. In yet another embodiment, the first primer comprises or consists of the oligonucleotide sequence of SEQ ID NO:6, the second primer comprises or consists of the oligonucleotide sequence of SEQ ID NO:18, and the detectable probe comprises or consists of the oligonucleotide sequence of SEQ ID NO:26.

[0021] In one embodiment, a primer set for amplification of a SARS-CoV-2 target comprises a first primer comprising or consisting of a first oligonucleotide sequence selected from the group consisting of SEQ ID NOs: 1-6, 27-31, and 40, and a second primer comprising or consisting of a second oligonucleotide sequence selected from the group consisting of SEQ ID NOs: 7-20, and 41.

[0022] In one embodiment, a primer set for amplification of a SARS-CoV-2 target comprises a first primer comprising or consisting of a first oligonucleotide sequence selected from the group consisting of SEQ ID NOs: 1-3 and 27-31, and a second primer comprising or consisting of a second oligonucleotide sequence selected from the group consisting of SEQ ID NOs: 7-14. In another embodiment, a primer set for amplification of a SARS-CoV-2 target comprises a first primer comprising or consisting of a first oligonucleotide sequence selected from the group consisting of SEQ ID NOs: 4-6, or a complement thereof, and a second primer comprising or consisting of a second oligonucleotide sequence selected from the group consisting of SEQ ID NOs: 15-20, or a complement thereof. In another embodiment, a primer set for amplification of a SARS-CoV-2 target comprises a first primer comprising or consisting of the oligonucleotide sequence of SEQ ID NO: 1, and a second primer comprising or consisting of the oligonucleotide sequence of SEQ ID NO: 7. In another embodiment, the first primer comprises or consists of an oligonucleotide sequence selected from the group consisting of SEQ ID NOs: 27 and 30, and the second primer comprises or consists of the oligonucleotide sequence of SEQ ID NO: 7. In another embodiment, the first primer comprises or consists of the oligonucleotide sequence of SEQ ID NO: 5, and the second primer comprises or consists of the oligonucleotide sequence of SEQ ID NO: 15. In yet another embodiment, the first primer comprises or consists of the oligonucleotide sequence of SEQ ID NO: 6, and the second primer comprises or consists of the oligonucleotide sequence of SEQ ID NO: 18. In yet another embodiment, the first primer comprises or consists of the oligonucleotide sequence of SEQ ID NO: 40, and the second primer comprises or consists of the oligonucleotide sequence of SEQ ID NO: 41.

[0023] In another embodiment, the primer set for amplification of a SARS-CoV-2 target comprises a plurality of first primers, a plurality of second primers, and a plurality of detectable probes, wherein the plurality of first primers is a combination of a first primer comprising an oligonucleotide sequence selected from the group consisting of SEQ ID NOs: 1 and 27 and a first primer comprising the oligonucleotide sequence of SEQ ID NO: 5; the plurality of second primers is a combination of a second primer comprising the oligonucleotide sequence of SEQ ID NO: 7 and a second primer comprising the oligonucleotide sequence of SEQ ID NO: 15; and the plurality of detectable probes is a combination of an oligonucleotide probe comprising the oligonucleotide sequence of SEQ ID NO: 21 and an oligonucleotide probe comprising an oligonucleotide sequence selected from the group consisting of SEQ ID NOs: 25 and 32. In yet another embodiment, the plurality of first primers is a combination of a first primer comprising an oligonucleotide sequence selected from the group consisting of SEQ ID NOs: 1 and 27, a first primer comprising an oligonucleotide sequence of SEQ ID NO: 5, and a first primer comprising an oligonucleotide sequence of SEQ ID NO: 6; the plurality of second primers is a combination of a second primer comprising an oligonucleotide sequence of SEQ ID NO: 7, a second primer comprising an oligonucleotide sequence of SEQ ID NO: 15, and a second primer comprising an oligonucleotide sequence of SEQ ID NO: 18; and the plurality of detectable probes is a combination of an oligonucleotide probe comprising an oligonucleotide sequence of SEQ ID NO: 21, an oligonucleotide probe comprising an oligonucleotide sequence selected from the group consisting of SEQ ID NOs: 25 and 32, and an oligonucleotide probe comprising an oligonucleotide sequence of SEQ ID NO: 26.In yet another embodiment, the plurality of first primers is a combination of a first primer comprising the oligonucleotide sequence of SEQ ID NO: 27 and a first primer comprising the oligonucleotide sequence of SEQ ID NO: 40, the plurality of second primers is a combination of a second primer comprising the oligonucleotide sequence of SEQ ID NO: 7 and a second primer comprising the oligonucleotide sequence of SEQ ID NO: 41, and the plurality of detectable probes is a combination of an oligonucleotide probe comprising the oligonucleotide sequence of SEQ ID NO: 42 and an oligonucleotide probe comprising the oligonucleotide sequence of SEQ ID NO: 43.

[0024] In another aspect, a method for simultaneously detecting SARS-CoV-2, influenza A, and influenza B in a sample, comprising: performing an amplification step, if SARS-CoV-2, and / or influenza A, and / or influenza B are present in the sample, comprising contacting the sample with a first primer set, a second primer set, and a third primer set to produce one or more amplification products, wherein the first primer set produces an amplification product if SARS-CoV-2 is present in the sample, the second primer set produces an amplification product if influenza A is present in the sample, and the third primer set produces an amplification product if influenza B is present in the sample. performing a hybridizing step comprising contacting the amplification product(s) with three or more detectable probes, wherein the three or more detectable probes comprise at least one probe specific for an amplification product of each of a first primer set, a second primer set, and a third primer set, and detecting the presence or absence of the amplification product, wherein the presence of the amplification product indicates the presence of SARS-CoV-2, influenza A, and / or influenza B in the sample, and the absence of the amplification product indicates the absence of SARS-CoV-2, influenza A, and / or influenza B in the sample. In some embodiments, the method further comprises providing a fourth primer set and a fourth detectable probe. wherein the fourth primer set is capable of producing an amplification product when SARS-CoV-2 or other coronavirus target nucleic acid from subgenus Sarbecovirus is present in the sample and the detectable fourth probe detects the presence or absence of the amplification product, the presence of the amplification product indicating the presence of SARS-CoV-2 or other coronavirus target nucleic acid from subgenus Sarbecovirus in the sample.In another embodiment, the fourth primer set is capable of producing an amplification product when SARS-CoV-2 is present in the sample and the detectable fourth probe detects the presence or absence of the amplification product, the presence of which indicates the presence of SARS-CoV-2 in the sample.

[0025] In another embodiment, the first primer set used in the method(s) comprises a forward primer comprising or consisting of an oligonucleotide sequence selected from the group consisting of SEQ ID NOs: 1-3 and 27-31 and a reverse primer comprising or consisting of an oligonucleotide sequence selected from the group consisting of SEQ ID NOs: 7-14; the second primer set comprises a forward primer comprising or consisting of the oligonucleotide sequence of SEQ ID NO: 33 and a reverse primer comprising or consisting of the oligonucleotide sequence of SEQ ID NO: 34; the third primer set comprises a forward primer comprising or consisting of the oligonucleotide sequence of SEQ ID NO: 36 and a reverse primer comprising or consisting of the oligonucleotide sequence of SEQ ID NO: 37; the first detectable probe comprises or consists of an oligonucleotide sequence selected from the group consisting of SEQ ID NOs: 21-23 and 42; the second detectable probe comprises or consists of the oligonucleotide sequence of SEQ ID NO: 35 or 44; and the third detectable probe comprises or consists of the oligonucleotide sequence of SEQ ID NO: 38 or 45. In another embodiment, the method further comprises providing a fourth primer set that produces an amplification product when SARS-CoV-2 or SARS-CoV-2 and other coronavirus target nucleic acids from the subgenus Sarbecovirus are present in the sample. In a specific embodiment, the fourth primer set comprises a forward primer comprising or consisting of an oligonucleotide sequence selected from the group consisting of SEQ ID NOs: 4-6 and 40 and a reverse primer comprising or consisting of an oligonucleotide sequence selected from the group consisting of SEQ ID NOs: 15-20 and 41, and the fourth detectable probe comprises or consists of an oligonucleotide sequence selected from the group consisting of SEQ ID NOs: 24-26, 32 and 43.In some embodiments, the fourth primer set comprises a forward primer comprising an oligonucleotide sequence selected from the group consisting of SEQ ID NOs: 4-6 and a reverse primer comprising an oligonucleotide sequence selected from the group consisting of SEQ ID NOs: 15-20, and the fourth detectable probe comprises an oligonucleotide sequence selected from the group consisting of SEQ ID NOs: 24-26 and 32. In some embodiments, a fourth primer set that produces a fourth amplification product when a SARS-CoV-2 target nucleic acid is present in a sample comprises a forward primer comprising the oligonucleotide sequence of SEQ ID NO: 40 and a reverse primer comprising the oligonucleotide sequence of SEQ ID NO: 41, and the fourth detectable probe for detecting the amplification product comprises the oligonucleotide sequence of SEQ ID NO: 43.

[0026] In one embodiment, a first primer set that generates an amplification product when SARS-CoV-2 is present in a sample comprises a forward primer that comprises or consists of the oligonucleotide sequence of SEQ ID NO: 27 and a reverse primer that comprises or consists of the oligonucleotide sequence of SEQ ID NO: 7, and a first detectable probe for detecting the amplification product comprises or consists of the oligonucleotide sequence of SEQ ID NO: 21 or 42. In another embodiment, a fourth primer set that generates an amplification product when SARS-CoV-2 or other coronavirus target nucleic acids from SARS-CoV-2 and the subgenus Sarbecovirus are present in a sample comprises a forward primer that comprises or consists of the oligonucleotide sequence of SEQ ID NO: 5 or 40 and a reverse primer that comprises or consists of the oligonucleotide sequence of SEQ ID NO: 15 or 41, and a fourth detectable probe for detecting the amplification product comprises or consists of the oligonucleotide sequence of SEQ ID NO: 32 or 43.

[0027] The present disclosure provides oligonucleotides comprising or consisting of a nucleotide sequence selected from SEQ ID NOs: 1-45 or its complement, the oligonucleotide having 100 or fewer nucleotides. Additionally, the present disclosure provides oligonucleotides comprising a nucleic acid having at least 70% sequence identity (e.g., at least 75%, 80%, 85%, 90%, or 95%) to one of SEQ ID NOs: 1-45 or its complement, the oligonucleotide having 100 or fewer nucleotides. Generally, these oligonucleotides can be primer nucleic acids, probe nucleic acids, etc., in these embodiments. In certain aspects, the oligonucleotide has 40 or fewer nucleotides (e.g., 35 or fewer nucleotides, 30 or fewer nucleotides, 25 or fewer nucleotides, 20 or fewer nucleotides, 15 or fewer nucleotides, etc.). In some aspects, the oligonucleotide comprises at least one modified nucleotide, e.g., to alter nucleic acid hybridization stability compared to unmodified nucleotides. The oligonucleotide optionally comprises at least one label and / or optionally at least one quencher moiety. In some aspects, the oligonucleotide comprises at least one conservatively modified mutation. "Conservatively modified variations" or simply "conservative variations" of a particular nucleic acid sequence refer to nucleic acids that encode identical or essentially identical amino acid sequences, or, where the nucleic acid does not encode an amino acid sequence, essentially identical sequences. Those of skill in the art will recognize that individual substitutions, deletions, or additions that alter, add, or delete a single nucleotide or a small percentage of nucleotides (typically less than 5%, more typically less than 4%, 2%, or 1%) in an encoded sequence are "conservatively modified variations" where the alteration results in the deletion of an amino acid, the addition of an amino acid, or the substitution of an amino acid with a chemically similar amino acid.

[0028] In one embodiment, amplification can use a polymerase enzyme with 5' to 3' nuclease activity. Thus, the donor fluorescent moiety and the acceptor moiety, e.g., a quencher, can be within 5 to 20 nucleotides (e.g., 8 or 10 nucleotides) of each other along the length of the probe. In other embodiments, the probe contains a nucleic acid sequence that allows for the formation of a secondary structure. The formation of such a secondary structure can result in spatial proximity between the first fluorescent moiety and the second fluorescent moiety. According to this method, the second fluorescent moiety on the probe can be a quencher.

[0029] In one embodiment, specific SARS-CoV-2, influenza A, and / or influenza B probes can be labeled with a fluorescent dye that acts as a reporter. The probe can also have a second dye that acts as a quencher. The reporter dye is measured at a defined wavelength, thus allowing for detection and discrimination of amplified SARS-CoV-2, influenza A, and / or influenza B targets. The fluorescent signal of the intact probe is suppressed by the quencher dye. During the PCR amplification step, hybridization of the probe to a specific single-stranded DNA template results in cleavage by the 5' to 3' nuclease activity of the DNA polymerase, separating the reporter and quencher dyes and generating a fluorescent signal. With each PCR cycle, the amount of cleaved probe increases, resulting in a concomitant increase in the cumulative signal of the reporter dye. Optionally, one or more additional probes (e.g., internal reference controls or other target probes (e.g., other viral nucleic acids)) can also be labeled with a reporter fluorescent dye that is unique and distinct from the fluorescent dye label associated with the SARS-CoV-2, influenza A, and / or influenza B probe. In such cases, simultaneous detection and discrimination of the amplified SARS-CoV-2, influenza A, and / or influenza B target and one or more additional probes is possible because the specific reporter dye is measured at a defined wavelength.

[0030] The present disclosure also provides methods for detecting the presence or absence of SARS-CoV-2 or SARS-CoV-2 nucleic acids in a biological sample from an individual. The present disclosure also provides methods for detecting the presence or absence of SARS-CoV-2, influenza A and / or influenza B, or SARS-CoV-2, influenza A and / or influenza B nucleic acid(s) in a biological sample from an individual. These methods can be used to detect the presence or absence of SARS-CoV-2 or SARS-CoV-2, influenza A and / or influenza B nucleic acid(s) in nasopharyngeal (NSP) and oropharyngeal swab samples for use in diagnostic testing. Furthermore, the same tests can be used by those skilled in the art to evaluate other sample types for the detection of SARS-CoV-2 or SARS-CoV-2, influenza A and / or influenza B nucleic acid(s). Such methods generally involve performing a reverse transcription step and at least one cycling step that includes an amplification step and a dye-binding step. Typically, the amplification step involves contacting the sample with multiple pairs of oligonucleotide primers that generate one or more amplification products if the nucleic acid molecule is present in the sample, and the dye-binding step involves contacting the amplification product with a double-stranded DNA-binding dye. Such methods also include detecting the presence or absence of binding of the double-stranded DNA-binding dye to the amplification product, where the presence of binding indicates the presence of SARS-CoV-2 or SARS-CoV-2, influenza A and / or influenza B nucleic acid(s) in the sample, and the absence of binding indicates the absence of SARS-CoV-2 or SARS-CoV-2, influenza A and / or influenza B nucleic acid(s) in the sample. A representative double-stranded DNA-binding dye is ethidium bromide. Other nucleic acid-binding dyes include DAPI, Hoechst dyes, PicoGreen®, RiboGreen®, OliGreen®, and cyanine dyes such as YO-YO® and SYBR® Green.Additionally, such methods may also include determining the melting temperature between the amplification product and a double-stranded DNA binding dye, which confirms the presence or absence of SARS-CoV-2 or SARS-CoV-2, influenza A and / or influenza B nucleic acid(s).

[0031] In a further embodiment, a kit is provided for detecting one or more nucleic acids of SARS-CoV-2. The kit may include one or more primer sets specific for amplifying gene targets; and one or more detectable oligonucleotide probes specific for detecting the amplification products. In another embodiment, a kit is provided for simultaneously detecting one or more nucleic acids of SARS-CoV-2, one or more nucleic acids of influenza A, and one or more nucleic acids of influenza B. The kit may include one or more primer sets specific for amplifying SARS-CoV-2 gene targets, influenza A gene targets, and influenza B gene targets, and one or more detectable oligonucleotide probes specific for detecting the amplification products of SARS-CoV-2, influenza A, and influenza B.

[0032] In one embodiment, the kit can include probes that are already labeled with donor and corresponding acceptor moieties, such as other fluorescent moieties or dark quenchers, or can include fluorophore moieties for labeling the probes. The kit can also further include nucleoside triphosphates, a nucleic acid polymerase, and buffers necessary for nucleic acid polymerase function. The kit can also include inserts and instructions for using the primers, probes, and fluorophore moieties to detect the presence or absence of SARS-CoV-2 nucleic acid or SARS-CoV-2, influenza A, and / or influenza B nucleic acid(s) in a sample.

[0033] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present subject matter, suitable methods and materials are described below. Additionally, the materials, methods, and examples are illustrative only and are not intended to be limiting.

[0034] The details of one or more embodiments of the invention are set forth in the accompanying drawings and the description below. Other features, objects, and advantages of the invention will be apparent from the drawings and detailed description, as well as the claims. [Brief explanation of the drawings]

[0035] [Figure 1] Figure 1 shows the genomic organization of SARS-CoV-2 (herein designated Wuhan-Hu-1) and SAR-CoV, as well as the location of the target regions of the SARS-CoV-2 primers and probes of the present invention. E: envelope protein gene; M: membrane protein gene; N: nucleocapsid protein gene; ORF1a / b: ORFs of nonstructural genes; S: spike protein gene; the numbers below the amplicons are the genomic locations according to Wuhan-Hu-1, GenBank MN908947. [Figure 2] Figure 2 shows PCR amplification curves from an experiment in which primers and probes for the nCoV1 assay were tested against various concentrations of linearized SARS-CoV-2 DNA template. [Figure 3] Figure 3 shows PCR amplification curves from an experiment in which primers and probes for the nCoV1 assay were tested against various concentrations of SARS-CoV-2 RNA transcripts. [Figure 4] Figure 4 shows PCR amplification curves from experiments in which primers and probes for the Pan-Sarbeco-2 assay were tested against various concentrations of linearized SARS-CoV-2 DNA template. [Figure 5]Figure 5 shows growth curves (left) and Ct charts (right) plotting the dynamic range over the indicated levels (1e+8 to 1e+1) tested using synthetic in vitro transcripts in the multiplex PCR test described in Example 5 for the nCoV1 assay (top) and the whole sarbecovirus-1 assay (bottom). [Figure 6] FIG. 6 shows a summary of the limit of detection (LOD) data from the experimental data shown in FIG. [Figure 7] Figure 7 shows amplification curves generated from SARS-CoV-2 testing using the indicated levels of isolated genomic RNA from patient samples diluted in specimen diluent (SD) in a multiplex PCR test that included an nCoV1 assay (left) and a whole sarbecovirus-1 assay (right). [Figure 8] Figure 8 shows amplification curves generated from SARS-CoV-2 testing using the indicated levels of isolated genomic RNA from patient samples diluted in nasopharyngeal specimen eluate (NSP) in a multiplex PCR test including the nCoV1 assay (left) and the whole sarbecovirus-1 assay (right). [Figure 9A-B] Figure 9A shows the genomic organization of SARS-CoV-2 (herein designated Wuhan-Hu-1) and SARS-CoV, and the locations of the target regions for the SARS-CoV-2 primers and probes in the SARS-CoV-2 & Influenza A / B assay. Figure 9B shows the locations of the target regions for the Influenza A primers and probes in the SARS-CoV-2 & Influenza A / B assay. [Figure 9C] Figure 9C shows the location of the target regions for the influenza B primers and probes for the SARS-CoV-2 & influenza A / B assay. [Figure 10] Figure 10 shows amplification curves in the multiplex PCR test described in Example 11 for the FluA assay (top, filter = 1) and the nCoV1 assay (bottom, filter = 2) across the indicated levels (1e+9 to 5e+0) tested using synthetic in vitro transcripts. [Figure 11]FIG. 11 shows amplification curves in the multiplex PCR test described in Example 11 for the whole sarbecovirus-1 assay (top, filter=3) and FluB assay (bottom, filter=4) across the indicated levels (1e+9 to 5e+0) tested using synthetic in vitro transcripts. [Figure 12] FIG. 12 shows a composite linearity plot (Ct chart plotting the dynamic range) of the amplification curves shown in FIGS. [Figure 13] FIG. 13 shows the amplification curves in a multiplex PCR test in a simulated nasopharyngeal matrix, as described in Example 12. DETAILED DESCRIPTION OF THE INVENTION

[0036] Detailed Description of the Invention Diagnosing SARS-CoV-2 infection by nucleic acid amplification provides a method for rapid, accurate, reliable, specific, and sensitive detection of viral infection. A real-time reverse transcriptase PCR assay for detecting SARS-CoV-2 in nonbiological or biological samples is described herein. Primers and probes for detecting SARS-CoV-2 are provided, as are articles of manufacture or kits containing such primers and probes. The increased specificity and sensitivity of real-time PCR for detecting SARS-CoV-2 compared to other methods, as well as improved features of real-time PCR, including sample containment and real-time detection of amplification products, make the implementation of this technology for routine diagnosis of SARS-CoV-2 infection feasible in clinical laboratories. Furthermore, this technology can be used for in vitro diagnosis and prognosis. This SARS-CoV-2 detection assay can also be multiplexed in parallel with other assays for the detection of other nucleic acids, such as influenza virus, SARS-CoV, and MERS-CoV.

[0037] Furthermore, simultaneous diagnosis of SARS-CoV-2, influenza A, and influenza B infections by nucleic acid amplification provides a method for rapid, accurate, reliable, specific, and sensitive detection and differentiation of these respiratory viral infections. Real-time reverse transcriptase PCR assays for detecting and differentiating SARS-CoV-2, influenza A, and influenza B in non-biological or biological samples are described herein. Primers and probes for detecting SARS-CoV-2, influenza A, and influenza B are provided, as are articles of manufacture or kits containing such primers and probes. The increased specificity and sensitivity of real-time PCR for the detection of SARS-CoV-2, influenza A, and influenza B compared with other methods, as well as improved features of real-time PCR, including sample containment and real-time detection of amplification products, make the implementation of this technology for routine diagnosis of SARS-CoV-2, influenza A, and influenza B infections feasible in clinical laboratories. Furthermore, this technology can be used for in vitro diagnosis and prognosis. This SARS-CoV-2 detection multiplex assay can also be further multiplexed in parallel with other assays for the detection of other viral targets, such as influenza C virus, influenza D virus, SARS-CoV, or MERS-CoV.

[0038] The SARS-CoV-2 genome is a 29,903-base-long, positive-sense, single-stranded RNA molecule (GenBank accession number MN908947) with the following gene order (5' to 3'): replicase ORF1ab (21,291 bases containing 16 predicted nonstructural proteins essential for viral replication and assembly), spike (S gene, 3,822 bases encoding the spike protein involved in binding to cellular receptors), ORF3ab (828 bases long), envelope (E gene, 228 bases encoding the envelope protein), membrane (M gene, 669 bases encoding the membrane protein), and nucleocapsid (N gene, 1260 bases encoding the nucleocapsid protein that forms a complex with the genomic RNA). Additionally, there is a 265-base noncoding region at the 5' end and a 229-base noncoding region at the 3' end.

[0039] The influenza A genome is a 13,588-base-long, segmented, negative-sense, single-stranded RNA molecule (see www.ncbi.nlm.nih.gov / genomes / FLU / FLU.html). Depending on the strain, the genome is composed of eight segments encoding 10–14 genes. From longest to shortest, the segments and their encoded genes are as follows: Segment 1 (RNA polymerase subunit PB2); Segment 2 (RNA polymerase subunit PB1 and PB1-F2 proteins); Segment 3 (RNA polymerase subunit PA and PA-X proteins); Segment 4 (hemagglutinin); Segment 5 (nucleoprotein); Segment 6 (neuraminidase); Segment 7 (matrix protein M1 and matrix protein M2); and Segment 8 (nonstructural proteins NS1 and NEP). Hemagglutinin and neuraminidase are large proteins found on the exterior of influenza virions. Hemagglutinin (HA) is responsible for binding of influenza virus particles to target cells and for the viral genome to enter the cells. Neuraminidase (NA) catalyzes the release of virions from infected cells. There are 16 subtypes of HA and 9 subtypes of NA, but only H1, H2, H3 and N1, N2 are commonly found in humans.

[0040] The influenza B genome, like the influenza A genome, is a 14,548-base-long, eight-segment, negative-sense, single-stranded RNA molecule. The influenza B genome is highly similar to that of influenza A, with a few exceptions. From longest to shortest, the segments and their encoded genes are as follows: segment 1 (RNA polymerase subunit PB2); segment 2 (RNA polymerase subunit PB1 protein); segment 3 (RNA polymerase subunit PA); segment 4 (hemagglutinin); segment 5 (nucleoprotein); segment 6 (neuraminidase and matrix protein NB); segment 7 (matrix protein M1 and membrane protein BM2); and segment 8 (nonstructural proteins NS1 and NEP). Influenza B is less prevalent in humans than influenza A, but disproportionately affects children and adolescents.

[0041] The present disclosure includes oligonucleotide primers and fluorescently labeled hydrolysis probes that hybridize to the SARS-CoV-2 genome (e.g., the ORF1ab gene and / or the E gene) for specific identification of SARS-CoV-2, for example, using TaqMan® amplification and detection technology. The oligonucleotides specifically hybridize to the ORF1ab gene and / or the E gene. Having oligonucleotides that hybridize to multiple locations within the genome is advantageous for improving sensitivity compared to targeting single-copy loci.

[0042] The disclosed methods may include performing a reverse transcription step and at least one cycling step involving amplifying one or more portions of a nucleic acid molecule gene target from a sample using one or more primer pairs. As used herein, "SARS-CoV-2 primer(s)" refers to oligonucleotide primers that specifically anneal to nucleic acid sequences found in the SARS-CoV-2 genome and initiate DNA synthesis therefrom under appropriate conditions to produce respective amplification products. Examples of nucleic acid sequences found in the SARS-CoV-2 genome include nucleic acids within the ORF1ab, S, ORF3ab, E, M, and N genes, as well as other predicted ORF regions. Each of the SARS-CoV-2 primers contemplated anneals to a target region such that at least a portion of each amplification product contains a nucleic acid sequence corresponding to the target. If one or more nucleic acids are present in the sample, one or more amplification products will be generated; therefore, the presence of one or more amplification products indicates the presence of SARS-CoV-2 in the sample. The amplification products must contain nucleic acid sequences complementary to one or more detectable probes for SARS-CoV-2. As used herein, "SARS-CoV-2 probe(s)" refers to oligonucleotide probes that specifically anneal to nucleic acids found in the SARS-CoV-2 genome. Each cycling step includes an amplification step, a hybridization step, and a detection step, in which the sample is contacted with one or more detectable SARS-CoV-2 probes to detect the presence or absence of SARS-CoV-2 in the sample.

[0043] Similarly, the terms "influenza A primer(s)" and "influenza B primer(s)," as used herein, refer to oligonucleotide primers that specifically anneal to nucleic acid sequences found in the influenza A genome and influenza B genome, respectively, and initiate DNA synthesis therefrom under appropriate conditions to produce respective amplification products. The terms "influenza A probe(s)" and "influenza B probe(s)," as used herein, refer to oligonucleotide probes that specifically anneal to nucleic acid sequences found in the SARS-CoV-2 genome and allow detection of the respective target amplification products.

[0044] As used herein, the term "amplifying" refers to the process of synthesizing a nucleic acid molecule complementary to one or both strands of a template nucleic acid molecule (e.g., a nucleic acid molecule from the SARS-CoV-2 genome). Amplifying a nucleic acid molecule typically involves denaturing the template nucleic acid, annealing primers to the template nucleic acid at a temperature below the melting temperature of the primers, and enzymatically extending from the primers to generate an amplification product. Amplification typically requires the presence of deoxyribonucleoside triphosphates, a DNA polymerase enzyme (e.g., Platinum® Taq), and an appropriate buffer and / or cofactors (e.g., MgCl and / or KCl) for optimal activity of the polymerase enzyme.

[0045] The term "primer," as used herein, is known to those skilled in the art and refers to oligomeric compounds, primarily oligonucleotides, but also modified oligonucleotides that are capable of "priming" DNA synthesis by a template-dependent DNA polymerase, i.e., the 3' end of the oligonucleotide provides a free 3'-OH group to which a further "nucleotide" can be attached by a template-dependent DNA polymerase that establishes a 3' to 5' phosphodiester bond, thereby using a deoxynucleoside triphosphate and thereby releasing pyrophosphate.

[0046] The term "hybridizing" refers to the annealing of one or more probes to an amplification product. "Hybridization conditions" typically include a temperature below the melting temperature of the probe, but which avoids non-specific hybridization of the probe.

[0047] The term "5' to 3' nuclease activity" refers to the activity of a nucleic acid polymerase typically associated with nucleic acid chain synthesis whereby nucleotides are removed from the 5' end of a nucleic acid chain.

[0048] The term "thermostable polymerase" refers to a polymerase enzyme that is thermostable, i.e., that catalyzes the formation of primer extension products complementary to a template and does not irreversibly denature when exposed to elevated temperatures for the time required to effect denaturation of the double-stranded template nucleic acid. Generally, synthesis is initiated at the 3' end of each primer and proceeds in the 5' to 3' direction along the template strand. Thermostable polymerases have been isolated from, for example, Thermus flavus, T. ruber, T. thermophilus, T. aquaticus, T. lacteus, T. rubens, Bacillus stearothermophilus, and Methanothermus fervidus. Nevertheless, polymerases that are not thermostable can also be used in PCR assays, provided the enzyme is replenished as needed.

[0049] The term "complement thereof" refers to a nucleic acid that is the same length as and exactly complementary to a given nucleic acid.

[0050] The terms "extension" or "lengthening" when used with respect to nucleic acids refer to when additional nucleotides (or other similar molecules) are incorporated into a nucleic acid. For example, a nucleic acid is optionally extended by a nucleotide-incorporating biocatalyst, e.g., by a polymerase, which typically adds a nucleotide to the 3' end of the nucleic acid.

[0051] The terms "identical" or percent "identity" in the context of two or more nucleic acid sequences refer to two or more sequences or subsequences that have the same or a specified percentage of identical nucleotides when compared and aligned for maximum correspondence as determined, for example, using one of the sequence comparison algorithms available to those of skill in the art or by visual inspection. Exemplary algorithms suitable for determining percent sequence identity and sequence similarity are the BLAST programs, see, e.g., Altschul et al. (1990) "Basic local alignment search tool," J. Mol. Biol. 215:403-410; Gish et al. (1993) "Identification of protein coding regions by database similarity search," Nature Genet. 3:266-272; Madden et al. (1996) "Applications of network BLAST server," Meth. Enzymol. 266:131-141; Altschul et al. (1997) "Gapped BLAST and PSI-BLAST: a new generation of protein database search programs," Nucleic Acids Res. 25:3389-3402; and Zhang et al. (1997) "PowerBLAST: A new network BLAST application for interactive or automated sequence analysis and annotation," Genome Res. 7:649-656.

[0052] "Modified nucleotides" in the context of oligonucleotides refers to alterations in which at least one nucleotide of the oligonucleotide sequence is replaced with a different nucleotide that provides desired properties to the oligonucleotide. Exemplary modified nucleotides that can be substituted in the oligonucleotides described herein include, for example, t-butylbenzyl, C5-methyl-dC, C5-ethyl-dC, C5-methyl-dU, C5-ethyl-dU, 2,6-diaminopurine, C5-propynyl-dC, C5-propynyl-dU, C7-propynyl-dA, C7-propynyl-dG, C5-propargylamino-dC, C5-propargylamino-dU, C7-propargylamino-dA, C7-propargylamino-dG, 7-deaza-2-deoxy-xanthosine, pyrazolopyrimidine analogs, pseudo-dU, nitropyrrole, nitroindole, 2'-0-methylribo-U, 2'-0-methylribo-C, N4-ethyl-dC, N6-methyl-dA, etc. Many other modified nucleotides that can be substituted in oligonucleotides are mentioned herein or known in the art. In certain embodiments, modified nucleotide substitutions modify the melting temperature (Tm) of the oligonucleotide compared to the melting temperature of the corresponding unmodified oligonucleotide.Furthermore, in some embodiments, certain modified nucleotide substitutions can reduce non-specific nucleic acid amplification (e.g., minimize primer-dimer formation, etc.), increase the yield of the intended target amplicon, etc. Examples of these types of nucleic acid modifications are described, for example, in U.S. Patent No. 6,001,611.Other modified nucleotide substitutions can change the stability of oligonucleotides or provide other desirable characteristics.

[0053] SARS-CoV-2 Amplification and Detection The present disclosure provides methods for detecting SARS-CoV-2, for example, by amplifying a portion of a SARS-CoV-2 nucleic acid sequence. The nucleic acid sequence for SARS-CoV-2 is available (e.g., GenBank Accession No. MN908947). Specifically, primers and probes for amplifying and detecting SARS-CoV-2 nucleic acid molecular targets are provided by embodiments of the present disclosure.

[0054] For detection of SARS-CoV-2, primers and probes for amplifying SARS-CoV-2 are provided. SARS-CoV-2 nucleic acids other than those exemplified herein can also be used to detect SARS-CoV-2 in a sample. For example, functional variants can be evaluated for specificity and / or sensitivity by those skilled in the art using routine methods. Exemplary functional variants can include, for example, one or more deletions, insertions, and / or substitutions in the SARS-CoV-2 nucleic acids disclosed herein.

[0055] More specifically, embodiments of oligonucleotides include nucleic acids having a sequence selected from SEQ ID NOs: 1-32 and 39-43, substantially identical variants thereof having at least, e.g., 80%, 90%, or 95% sequence identity to one of SEQ ID NOs: 1-32 and 39-43, or complements and variants of SEQ ID NOs: 1-32 and 39-43, respectively. [Table 1] [Table 2] [Table 3]

[0056] In one embodiment, the SARS-CoV-2 primer and probe sets described above are used to provide for detection of SARS-CoV-2 in biological samples suspected of containing SARS-CoV-2 (Tables 1-3). The primer sets and probes can comprise or consist of primers and probes specific for SARS-CoV-2 nucleic acid sequences comprising or consisting of the nucleic acid sequences of SEQ ID NOS: 1-32 and 39-43. In another embodiment, the primers and probes for SARS-CoV-2 targets comprise or consist of functionally active variants of any of the primers and probes of SEQ ID NOS: 1-32 and 39-43.

[0057] Functionally active variants of any of the primers and / or probes of SEQ ID NOS: 1-32 and 39-43 can be identified by using the primers and / or probes in the disclosed methods. Functionally active variants of any of the primers and / or probes of SEQ ID NOS: 1-32 and 39-43 relate to primers and / or probes that provide similar or greater specificity and sensitivity in the described methods or kits compared to the respective sequences of SEQ ID NOS: 1-32 and 39-43.

[0058] A variant may vary from the sequences of SEQ ID NOS: 1-32 and 39-43 by the addition, deletion, or substitution of one or more nucleotides, such as the addition, deletion, or substitution of one or more nucleotides at the 5' and / or 3' ends of the sequences of SEQ ID NOS: 1-32 and 39-43, respectively. As detailed above, a primer (and / or probe) may be chemically modified, i.e., the primer and / or probe may contain modified nucleotides or non-nucleotide compounds. Thus, a probe (or primer) is a modified oligonucleotide. A "modified nucleotide" (or "nucleotide analog") differs from a natural "nucleotide" by some modification but still consists of a base or base-like compound, a pentofuranosyl sugar or pentofuranosyl sugar-like compound, a phosphate moiety or phosphate-like moiety, or a combination thereof. For example, a "label" may be attached to the base portion of a "nucleotide" to obtain a "modified nucleotide." A natural base in a "nucleotide" may be replaced, for example, with a 7-deazapurine, thereby similarly obtaining a "modified nucleotide." The terms "modified nucleotide" or "nucleotide analog" are used interchangeably in this application. A "modified nucleoside" (or "nucleoside analog") differs from a naturally occurring nucleoside by some modification, as outlined above for "modified nucleotides" (or "nucleotide analogs").

[0059] Oligonucleotides, including modified oligonucleotides and oligonucleotide analogs, that amplify nucleic acid molecules encoding SARS-CoV-2 targets or nucleic acids encoding other portions of SARS-CoV-2 can be designed using computer programs such as OLIGO (Molecular Biology Insights Inc., Cascade, Colo.). Important design features for oligonucleotides used as amplification primers include, but are not limited to, appropriate size amplification product for ease of detection (e.g., by electrophoresis), similar melting temperatures for the members of the primer pair, and the length of each primer (i.e., primers must be long enough to anneal and initiate synthesis with sequence specificity, but not so long that fidelity is compromised during oligonucleotide synthesis). Typically, oligonucleotide primers are 8 to 50 nucleotides in length (e.g., 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, or 50 nucleotides in length).

[0060] In addition to a primer set, the method may use one or more probes to detect the presence or absence of SARS-CoV-2. The term "probe" refers to a synthetically or biologically produced nucleic acid (DNA or RNA) that, by design or selection, contains a specific nucleotide sequence that allows it to specifically (i.e., preferentially) hybridize to a "target nucleic acid" under a defined, predetermined stringency when SARS-CoV-2 (target) nucleic acid is present. A "probe" may also be referred to as a "detection probe," meaning that it detects a target nucleic acid.

[0061] In some embodiments, the disclosed SARS-CoV-2 probes can be labeled with at least one fluorescent label. In one embodiment, the SARS-CoV-2 probes can be labeled with a donor fluorescent moiety, e.g., a fluorescent dye, and a corresponding acceptor moiety, e.g., a quencher. In one embodiment, the probe comprises or consists of a fluorescent moiety, and the nucleic acid sequence comprises or consists of SEQ ID NOs: 21-26, 32, and 42-43.

[0062] Oligonucleotides used as probes can be designed in a similar manner to primer design. Embodiments can use a single probe or a pair of probes for detection of amplification products. Depending on the embodiment, the probe(s) used can include at least one label and / or at least one quencher moiety. Like primers, probes typically have similar melting temperatures, and the length of each probe must be sufficient for sequence-specific hybridization to occur, but not so long that fidelity is compromised during synthesis. Oligonucleotide probes are generally 15 to 40 (e.g., 16, 18, 20, 21, 22, 23, 24, or 25) nucleotides in length.

[0063] The constructs can include vectors, each containing one of the SARS-CoV-2 primer and probe nucleic acid molecules. The constructs can be used, for example, as a control template nucleic acid molecule. Suitable vectors for use are commercially available and / or produced by recombinant nucleic acid technology methods routine in the art. SARS-CoV-2 nucleic acid molecules can be obtained, for example, by chemical synthesis, direct cloning from SARS-CoV-2, or nucleic acid amplification.

[0064] Constructs suitable for use in the present methods typically include, in addition to a SARS-CoV-2 nucleic acid molecule (e.g., a nucleic acid molecule comprising one or more of the sequences of SEQ ID NOS: 1-32 and 39-43), a sequence encoding a selectable marker (e.g., an antibiotic resistance gene) for selection of the desired construct and / or transformant, and an origin of replication. The choice of vector system typically depends on several factors, including, but not limited to, host cell choice, replication efficiency, selectability, inducibility, and ease of recovery.

[0065] Constructs containing SARS-CoV-2 nucleic acid molecules can be propagated in host cells. As used herein, the term host cell is meant to include prokaryotic and eukaryotic organisms, such as yeast, plant, and animal cells. Prokaryotic hosts include Escherichia coli (E. coli), Salmonella typhimurium, Serratia marcescens, and Bacillus subtilis. Eukaryotic hosts include yeasts such as S. cerevisiae, S. pombe, and Pichia pastoris; mammalian cells such as COS cells or Chinese hamster ovary (CHO) cells; insect cells; and plant cells such as Arabidopsis thaliana and Nicotiana tabacum. The construct can be introduced into host cells using any of the techniques commonly known to those skilled in the art. For example, calcium phosphate precipitation, electroporation, heat shock, lipofection, microinjection, and viral-mediated nucleic acid transfer are common methods for introducing nucleic acids into host cells. Additionally, naked DNA can be delivered directly into cells (see, e.g., U.S. Patent Nos. 5,580,859 and 5,589,466).

[0066] Amplification and detection of influenza A and influenza B The present disclosure also provides methods for simultaneously detecting SARS-CoV-2, influenza A, and influenza B by amplifying a portion of a SARS-CoV-2 nucleic acid sequence, a portion of an influenza A nucleic acid sequence, and a portion of an influenza B nucleic acid sequence. Primers and probes for amplifying and detecting SARS-CoV-2 nucleic acid molecular targets are described in the previous section. Nucleic acid sequences for influenza A and influenza B are available under GenBank accession numbers KC781450 (A / Michigan / 01 / 2010(H1N1)) and KM654608 (B / Connecticut / Flu103 / 2013), respectively. Specifically, primers and probes for amplifying and detecting nucleic acid molecular targets for influenza A and influenza B are provided by embodiments of the present disclosure. Figures 9B and 9C show the locations targeted by influenza A primers and probes (SEQ ID NOS: 33-35 and 44 target the M1 and M2 genes of segment 7) and influenza B primers and probes (SEQ ID NOS: 36-38 and 45 target the NEP and NS1 genes of segment 8).

[0067] More specifically, embodiments of the oligonucleotides include nucleic acids having a sequence selected from, for influenza A, SEQ ID NOs: 33-35 and 44, a substantially identical variant thereof having at least, e.g., 80%, 90%, or 95% sequence identity to one of SEQ ID NOs: 33-35 and 44, or a complement of SEQ ID NOs: 33-35 and 44, and variants; or, for influenza B, SEQ ID NOs: 36-38 and 45, a substantially identical variant thereof having at least, e.g., 80%, 90%, or 95% sequence identity to one of SEQ ID NOs: 36-38 and 45, or a complement of SEQ ID NOs: 36-38 and 45, and variants. [Table 4]

[0068] polymerase chain reaction (PCR) U.S. Patent Nos. 4,683,202, 4,683,195, 4,800,159, and 4,965,188 disclose conventional PCR techniques. PCR typically uses two oligonucleotide primers that bind to a selected nucleic acid template (e.g., DNA or RNA). In some embodiments, useful primers include oligonucleotides (e.g., SEQ ID NOS: 1-20, 27-31, 40-41) that can act as initiation points for nucleic acid synthesis within the described SARS-CoV-2 nucleic acid sequence. Primers can be purified from restriction digests by conventional methods or produced synthetically. While primers are preferably single-stranded for maximum efficiency in amplification, primers can also be double-stranded. Double-stranded primers are first denatured, i.e., treated to separate the strands. One method for denaturing double-stranded nucleic acids is by heating.

[0069] If the template nucleic acid is double-stranded, it is necessary to separate the two strands before it can be used as a template in PCR. Strand separation can be achieved by any suitable denaturing method, including physical, chemical, or enzymatic means. One method for separating nucleic acid strands involves heating the nucleic acid until it is predominantly denatured (e.g., greater than 50%, 60%, 70%, 80%, 90%, or 95% denatured). The heating conditions required to denature the template nucleic acid depend, for example, on the buffer salt concentration and the length and nucleotide composition of the nucleic acid to be denatured, but typically range from about 90°C to about 105°C, depending on reaction characteristics such as temperature and nucleic acid length. Denaturation is typically carried out for about 30 seconds to 4 minutes (e.g., 1 minute to 2 minutes 30 seconds, or 1.5 minutes).

[0070] Once the double-stranded template nucleic acid has been denatured by heat, the reaction mixture is cooled to a temperature that promotes annealing of each primer to its target sequence. The annealing temperature is typically about 35°C to about 65°C (e.g., about 40°C to about 60°C, about 45°C to about 50°C). The annealing time can be about 10 seconds to about 1 minute (e.g., about 20 seconds to about 50 seconds, about 30 seconds to about 40 seconds). The reaction mixture is then adjusted to a temperature that promotes or optimizes polymerase activity, i.e., a temperature sufficient to allow extension of the annealed primers to produce a product complementary to the template nucleic acid. The temperature must be sufficient to synthesize an extension product from each primer annealed to the nucleic acid template, but not so high as to denature the extension product from its complementary template (e.g., temperatures for extension generally range from about 40°C to about 80°C (e.g., about 50°C to about 70°C, about 60°C)). Extension times can be from about 10 seconds to about 5 minutes (e.g., about 30 seconds to about 4 minutes, about 1 minute to about 3 minutes, or about 1 minute 30 seconds to about 2 minutes).

[0071] The genomes of retroviruses or RNA viruses, such as SARS-CoV-2, as well as other flaviviruses, are composed of ribonucleic acid, or RNA. In such cases, the template nucleic acid, RNA, must first be transcribed into complementary DNA (cDNA) through the action of the enzyme reverse transcriptase. Using the RNA template and a short primer complementary to the 3' end of the RNA, reverse transcriptase directs the synthesis of first-strand cDNA, which can then be used directly as a template for the polymerase chain reaction.

[0072] PCR assays can use SARS-CoV-2 nucleic acids, such as RNA or DNA (cDNA). The template nucleic acid need not be purified and can be a minor fraction of a complex mixture, such as SARS-CoV-2 nucleic acids contained in human cells. SARS-CoV-2 nucleic acid molecules can be extracted from biological samples by routine techniques, such as those described in Diagnostic Molecular Microbiology: Principles and Applications (Persing et al. (eds), 1993, American Society for Microbiology, Washington, DC). Nucleic acids can be obtained from any number of sources, including plasmids or natural sources, including bacteria, yeast, viruses, organelles, or higher organisms such as plants or animals.

[0073] Oligonucleotide primers (e.g., SEQ ID NOS: 1-20, 27-31, 40-41) are combined with PCR reagents under reaction conditions conducive to primer extension. For example, a chain extension reaction typically contains 50 mM KCl, 10 mM Tris-HCl (pH 8.3), 15 mM MgCl, 0.001% (w / v) gelatin, 0.5-1.0 μg of denatured template DNA, 50 pmol of each oligonucleotide primer, 2.5 U of Taq polymerase, and 10% DMSO. The reaction typically contains 150-320 μM each of dATP, dCTP, dTTP, dGTP, or one or more analogs thereof.

[0074] The newly synthesized strands form double-stranded molecules that can be used in subsequent steps of the reaction. The strand separation, annealing, and extension steps can be repeated as many times as necessary to generate the desired amount of amplification product corresponding to the target SARS-CoV-2 nucleic acid molecule. The limiting factors in the reaction are the amount of primers, thermostable enzyme, and nucleoside triphosphates present in the reaction. The cycling steps (i.e., denaturation, annealing, and extension) are preferably repeated at least once. For detection applications, the number of cycling steps depends, for example, on the nature of the sample. If the sample is a complex mixture of nucleic acids, more cycling steps will be required to amplify the target sequence sufficiently for detection. Generally, the cycling steps are repeated at least about 20 times, but may be repeated 40, 60, or 100 times.

[0075] Fluorescence Resonance Energy Transfer (FRET) FRET technology (see, e.g., U.S. Pat. Nos. 4,996,143, 5,565,322, 5,849,489, and 6,162,603) is based on the concept that when a donor fluorescent moiety and a corresponding acceptor fluorescent moiety are positioned within a certain distance from each other, energy transfer occurs between the two fluorescent moieties, which can be visualized or otherwise detected and / or quantified. Typically, when excited by light irradiation of a suitable wavelength, the donor transfers energy to the acceptor. Typically, the acceptor re-emits the transferred energy in the form of light irradiation of a different wavelength. In certain systems, non-fluorescent energy can be transferred between the donor and acceptor moieties via a biomolecule that includes a substantially non-fluorescent donor moiety (see, e.g., U.S. Pat. No. 7,741,467).

[0076] In one example, an oligonucleotide probe contains a donor fluorescent moiety (e.g., HEX) and a corresponding quencher (e.g., BlackHole), which may or may not be fluorescent, that dissipates the transferred energy in forms other than light. The probe may contain a fluorescent donor moiety such that the fluorescent emission from the donor moiety is quenched by the acceptor moiety. When the probe is intact, energy transfer typically occurs between the donor and acceptor moieties, such that the fluorescent emission from the donor moiety is quenched by the acceptor moiety. During the extension step of the polymerase chain reaction, the probe bound to the amplification product is cleaved, for example, by the 5' to 3' nuclease activity of Taq polymerase, such that the fluorescent emission of the donor moiety is no longer quenched. Exemplary probes for this purpose are described, for example, in U.S. Patent Nos. 5,210,015, 5,994,056, and 6,171,785. Commonly used donor-acceptor pairs include the FAM-TAMRA pair. Commonly used quenchers are DABCYL and TAMRA. Commonly used dark quenchers include BlackHole Quenchers™ (BHQ), (Biosearch Technologies, Inc., Novato, California), Iowa. Black™ (Integrated DNA Tech., Inc., Coralville, IA), BlackBerry™ Quencher 650 (BBQ-650) (Berry & Assoc, Dexta, MI).

[0077] In another example, two oligonucleotide probes, each containing a fluorescent moiety, can hybridize to the amplification product at specific positions determined by the complementarity of the oligonucleotide probe to the SARS-CoV-2 target nucleic acid sequence. When the oligonucleotide probe hybridizes to the amplification product nucleic acid at the appropriate position, a FRET signal is generated. The hybridization temperature can range from about 35°C to about 65°C for about 10 seconds to about 1 minute.

[0078] Fluorescence analysis can be performed, for example, using a photon-counting epifluorescence microscope system (equipped with appropriate dichroic mirrors and filters to monitor a specific range of fluorescence emission), a photon-counting photomultiplier system, or a fluorometer. Excitation to initiate energy transfer or allow direct detection of the fluorophore can be performed using an argon ion laser, a high-intensity mercury (Hg) arc lamp, a xenon lamp, a fiber optic light source, or other high-intensity light source appropriately filtered for excitation of the desired range.

[0079] As used herein with respect to a donor moiety and a corresponding acceptor moiety, "corresponding" refers to an acceptor fluorescent moiety or dark quencher that has an absorbance spectrum that overlaps with the emission spectrum of the donor fluorescent moiety. The wavelength maximum of the emission spectrum of the acceptor fluorescent moiety must be at least 100 nm greater than the wavelength maximum of the excitation spectrum of the donor fluorescent moiety. Therefore, efficient non-irradiative energy transfer can occur between them.

[0080] Fluorescent donor moieties and corresponding acceptor moieties are generally selected for (a) efficient Förster energy transfer, (b) a large final Stokes shift (>100 nm), (c) emission shift as far as possible toward the red portion of the visible spectrum (>600 nm), and (d) emission shift to a wavelength higher than the Raman water fluorescence emission produced by excitation at the donor excitation wavelength. For example, a donor fluorescent moiety can be selected that has its maximum excitation wavelength near a laser line (e.g., helium-cadmium 442 nm or argon 488 nm), a high extinction coefficient, a high quantum yield, and good overlap of its fluorescence emission with the excitation spectrum of the corresponding acceptor fluorescent moiety. A corresponding acceptor fluorescent moiety can be selected that has a high extinction coefficient, a high quantum yield, good excitation overlap with the emission of the donor fluorescent moiety, and emission in the red portion of the visible spectrum (>600 nm).

[0081] Representative donor fluorescent moieties that can be used with various acceptor fluorescent moieties in FRET technology include fluorescein, Lucifer Yellow, B-phycoerythrin, 9-acridine isothiocyanate, Lucifer Yellow VS, 4-acetamido-4'-isothio-cyanatostilbene-2,2'-disulfonic acid, 7-diethylamino-3-(4'-isothiocyanatophenyl)-4-methylcoumarin, succinyl 1-pyrenebutyrate, and 4-acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic acid derivatives. Representative acceptor fluorescent moieties include LC Red 640, LC Red 705, Cy5, Cy5.5, Lissamine rhodamine B sulfonyl chloride, tetramethylrhodamine isothiocyanate, rhodamine x isothiocyanate, erythrosine isothiocyanate, fluorescein, diethylenetriamine pentaacetate, or other chelates of lanthanide ions (e.g., europium or terbium), depending on the donor fluorescent moiety used. Donor and acceptor fluorescent moieties can be obtained, for example, from Molecular Probes (Junction City, Oregon) or Sigma Chemical Co. (St. Louis, Missouri).

[0082] The donor and acceptor fluorescent moieties can be attached to the appropriate probe oligonucleotide via linker arms. The length of each linker arm is important because it affects the distance between the donor and acceptor fluorescent moieties. The length of the linker arm can be the distance in angstroms (Å) from the nucleotide base to the fluorescent moiety. Typically, the linker arm is about 10 Å to about 25 Å. The linker arm can be of the type described in WO 84 / 03285. WO 84 / 03285 also discloses methods for attaching the linker arm to a specific nucleotide base and for attaching the fluorescent moiety to the linker arm.

[0083] Acceptor fluorescent moieties such as LC Red 640 can be combined with oligonucleotides containing amino linkers (e.g., C6-aminophosphoramidites available from ABI (Foster City, CA) or Glen Research (Sterling, VA)) to produce, for example, LC Red 640-labeled oligonucleotides. Commonly used linkers for attaching donor fluorescent moieties such as fluorescein to oligonucleotides include thiourea linkers (derived from FITC, e.g., Fluorescein-CPG from Glen Research or ChemGene (Ashland, MA)), amide-linkers (derived from fluorescein-NHS-esters, such as CX-Fluorescein-CPG from BioGenex (San Ramon, CA)), or 3'-amino-CPG, which requires attachment of the fluorescein-NHS-ester after oligonucleotide synthesis.

[0084] Detection of amplification products The present disclosure provides a method for detecting the presence or absence of SARS-CoV-2 in a biological or non-biological sample. The provided method avoids problems of sample contamination, false negatives, and false positives. The method includes a reverse transcription step, at least one cycling step including amplifying a portion of a SARS-CoV-2 target nucleic acid molecule from the sample using one or more pairs of SARS-CoV-2 primers, and a FRET detection step. The multiple cycling steps are preferably performed in a thermocycler. The method can be performed using SARS-CoV-2 primers and probes to detect the presence of SARS-CoV-2, where detection of SARS-CoV-2 indicates the presence of SARS-CoV-2 in the sample.

[0085] As described herein, amplification products can be detected using labeled hybridization probes that utilize FRET technology. One FRET format utilizes TaqMan® technology to detect the presence or absence of amplification products, and therefore the presence or absence of SARS-CoV-2. TaqMan® technology utilizes, for example, a single-stranded hybridization probe labeled with one fluorescent dye (e.g., HEX) and one quencher (e.g., BHQ), which may or may not be fluorescent. When the first fluorescent moiety is excited with light of the appropriate wavelength, the absorbed energy is transferred to a second fluorescent moiety or dark quencher according to the principles of FRET. The second fluorescent moiety is typically a quencher molecule. During the annealing step of the PCR reaction, the labeled hybridization probe binds to the target DNA (i.e., the amplification product) and is subsequently degraded during the extension phase, for example, by the 5' to 3' nuclease activity of Taq polymerase. As a result, the fluorescent and quencher moieties are spatially separated from each other. As a result, upon excitation of the first fluorescent moiety in the absence of the quencher, fluorescent emission from the first fluorescent moiety can be detected. By way of example, the ABI PRISM® 7700 Sequence Detection System (Applied Biosystems) uses TaqMan® technology and is suitable for performing the methods described herein for detecting the presence or absence of SARS-CoV-2 in a sample.

[0086] Molecular beacons combined with FRET can also be used to detect the presence of amplification products using real-time PCR. Molecular beacon technology uses a hybridization probe labeled with a first fluorescent moiety and a second fluorescent moiety. The second fluorescent moiety is generally a quencher, and fluorescent labels are typically located at each end of the probe. Molecular beacon technology uses a probe oligonucleotide with a sequence that allows secondary structure formation (e.g., a hairpin). As a result of the formation of the secondary structure within the probe, both fluorescent moieties are spatially close together when the probe is in solution. After hybridization to the target nucleic acid (i.e., the amplification product), the secondary structure of the probe is disrupted, separating the fluorescent moieties from each other, allowing the emission of the first fluorescent moiety to be detected after excitation with light of an appropriate wavelength.

[0087] Another common form of FRET technology utilizes two hybridization probes. Each probe can be labeled with a different fluorescent moiety and is generally designed to hybridize in close proximity to one another within a target DNA molecule (e.g., an amplification product). A donor fluorescent moiety, e.g., fluorescein, is excited at 470 nm by the LightCycler® Instrument's light source. During FRET, the fluorescein transfers its energy to an acceptor fluorescent moiety, e.g., LightCycler®-Red 640 (LC Red 640) or LightCycler®-Red 705 (LC Red 705). The acceptor fluorescent moiety then emits light of a longer wavelength, which is detected by the LightCycler® Instrument's optical detection system. Efficient FRET can occur only when the fluorescent moieties are in direct local proximity and the emission spectrum of the donor fluorescent moiety overlaps with the absorption spectrum of the acceptor fluorescent moiety. The intensity of the emitted signal can be correlated with the number of original target DNA molecules (e.g., the number of SARS-CoV-2 genomes). If amplification of the SARS-CoV-2 target nucleic acid occurs and an amplification product is produced, the hybridization step results in a detectable signal based on FRET between the members of the probe pair.

[0088] Generally, the presence of FRET indicates the presence of SARS-CoV-2 in the sample, and the absence of FRET indicates the absence of SARS-CoV-2 in the sample. However, inadequate specimen collection, transport delays, improper transport conditions, or the use of certain collection swabs (calcium alginate or aluminum shafts) are all conditions that can affect the success and / or accuracy of the test results.

[0089] Representative biological samples that can be used to practice the present methods include, but are not limited to, respiratory specimens (nasopharyngeal and oropharyngeal swabs), urine, fecal specimens, blood specimens, plasma, skin swabs, wound swabs, blood cultures, and skin and soft tissue infections. Methods for collecting and storing biological samples are known to those of skill in the art. The biological sample may be processed (e.g., by nucleic acid extraction methods and / or kits known in the art) to release SARS-CoV-2 nucleic acids, or in some cases, the biological sample may be directly contacted with PCR reaction components and appropriate oligonucleotides.

[0090] Melting curve analysis is an additional step that can be included in the cycle profile. Melting curve analysis is based on the fact that DNA melts at a characteristic temperature called the melting temperature (Tm), which is defined as the temperature at which one half of a DNA duplex separates into single strands. The melting temperature of DNA depends primarily on its nucleotide composition. Thus, DNA molecules rich in G and C nucleotides have a higher Tm than DNA molecules rich in A and T nucleotides. By detecting the temperature at which the signal is lost, the probe's melting temperature can be determined. Similarly, by detecting the temperature at which the signal is generated, the probe's annealing temperature can be determined. The melting temperature(s) of a SARS-CoV-2 probe from a SARS-CoV-2 amplification product can confirm the presence or absence of SARS-CoV-2 in a sample.

[0091] During each thermocycler run, control samples can also be cycled. A positive control sample can amplify a target nucleic acid control template (other than the amplification product of the listed target gene) using, for example, control primers and a control probe. A positive control sample can also amplify, for example, a plasmid construct containing the target nucleic acid molecule. Such a plasmid control can be amplified internally (e.g., in the sample) or in a separate sample run alongside the patient sample using the same primers and probes used to detect the intended target. Such controls are indicators of the success or failure of the amplification, hybridization, and / or FRET reaction. Each thermocycler run can also include, for example, a negative control lacking target template DNA. The negative control can measure contamination, ensuring that the system and reagents do not produce false-positive signals. Thus, control reactions can easily determine, for example, the ability of primers to anneal with sequence specificity and initiate elongation, and the ability of probes to hybridize with sequence specificity and allow FRET to occur.

[0092] In one embodiment, the method includes a step to avoid contamination, for example, the enzymatic method utilizing uracil-DNA glycosylase described in U.S. Patent Nos. 5,035,996, 5,683,896, and 5,945,313 to reduce or eliminate contamination between one thermocycling run and the next.

[0093] This method can be carried out using conventional PCR combined with FRET technology. In one embodiment, a LightCycler® instrument is used. The following patent applications describe real-time PCR used with LightCycler® technology: WO 97 / 46707, WO 97 / 46714, and WO 97 / 46712.

[0094] The LightCycler® can be operated using a PC workstation and utilizes the Windows NT operating system. Signals from samples are acquired as the machine sequentially positions capillaries on the optical unit. The software can display the fluorescence signal in real time immediately after each measurement. Fluorescence acquisition times range from 10 to 100 milliseconds (msec). After each cycling step, a quantitative display of fluorescence versus cycle number can be continuously updated for all samples. The generated data can be saved for further analysis.

[0095] As an alternative to FRET, double-stranded DNA binding dyes, such as fluorescent DNA binding dyes (e.g., SYBR® Green or SYBR® Gold (Molecular Probes)), can be used to detect amplification products. Upon interaction with double-stranded nucleic acids, such fluorescent DNA binding dyes emit a fluorescent signal after excitation with light of an appropriate wavelength. Double-stranded DNA binding dyes, such as nucleic acid intercalating dyes, can also be used. When using double-stranded DNA binding dyes, a melting curve analysis is usually performed to confirm the presence of amplification products.

[0096] Those skilled in the art will appreciate that other nucleic acid or signal amplification methods may also be used, including, but not limited to, branched DNA signal amplification, loop-mediated isothermal amplification (LAMP), nucleic acid sequence-based amplification (NASBA), self-sustained sequence replication (3 SR), strand displacement amplification (SDA), or smart amplification process version 2 (SMAP 2).

[0097] It is understood that embodiments of the present disclosure are not limited by the configuration of one or more commercially available devices.

[0098] Manufactured Products / Kits Embodiments of the present disclosure further provide articles of manufacture, compositions, or kits for detecting SARS-CoV-2. The articles of manufacture may include primers and probes used to detect target SARS-CoV-2 genes, along with suitable packaging materials. Exemplary primers and probes for detecting SARS-CoV-2 may hybridize to SARS-CoV-2 target nucleic acid molecules. Additionally, the kits may also include appropriately packaged reagents and materials necessary for DNA immobilization, hybridization, and detection, such as solid supports, buffers, enzymes, and DNA standards. Methods for designing primers and probes are disclosed herein, and representative examples of primers and probes that amplify and hybridize to SARS-CoV-2 target nucleic acid molecules are provided.

[0099] The article of manufacture can also include one or more fluorescent moieties for labeling the probe, or can label the probe provided with the kit. For example, the article of manufacture can include donor and / or acceptor fluorescent moieties for labeling the SARS-CoV-2 probe. Examples of suitable FRET donor fluorescent moieties and corresponding acceptor fluorescent moieties are provided above.

[0100] The article of manufacture may also include a package insert or packaging label with instructions for using the SARS-CoV-2 primers and probes to detect SARS-CoV-2 in a sample. The article of manufacture may further include reagents (e.g., buffers, polymerase enzymes, cofactors, or agents to prevent contamination) for carrying out the methods disclosed herein. Such reagents may be specific to one of the commercially available instruments described herein.

[0101] Embodiments of the present disclosure are further described in the following examples, which do not limit the scope of the claimed invention. [Example]

[0102] The following examples and figures are provided to aid the understanding of the subject matter, the true scope of which is set forth in the appended claims. It is understood that modifications can be made in the procedures set forth without departing from the spirit of the invention.

[0103] Example 1: SARS-CoV-2 Assay Description A real-time reverse transcription polymerase chain reaction (RT-PCR) test was developed for the cobas® 6800 / 8800 System that enables the qualitative detection of nucleic acids from SARS-CoV-2 in nasopharyngeal (NSP) and oropharyngeal swab samples from patients who meet CDC clinical criteria. The assay detects: (i) a specific nucleic acid sequence from the nonstructural open reading frame (ORF1a / b) in the SARS-CoV-2 genome in one channel and (ii) a conserved sequence in the structural envelope (E) gene location common to all sarbecoviruses, including SARS-CoV-2, in a different channel. Results demonstrate the specific detection of SARS-CoV-2 RNA detectable in nasopharyngeal and oropharyngeal swab samples during the acute phase of infection.

[0104] Nucleic acids from patient samples and added RNA internal control molecules (identical to existing RNA QS reagents) are simultaneously extracted. Viral nucleic acids are released by adding proteinase and lysis reagents to the sample. The released nucleic acids bind to the silica surface of added magnetic glass particles. Unbound material and impurities, such as denatured proteins, cellular debris, and potential PCR inhibitors, are removed in subsequent wash steps, and the purified nucleic acids are eluted from the magnetic glass particles with a hot elution buffer.

[0105] Example 2: SARS CoV-2 Assay Design Strategy The diagnostic test was designed for the detection of the novel coronavirus (SARS-CoV-2), classified in the Coronaviridae family, Betacoronavirus genus, and Sarbecovirus subgenus (Lu et al., Lancet, 2020, (20)30251-8). This coronavirus is novel, and it is not known which regions undergo mutation or recombination. Given the lack of knowledge about this virus, a single-analyte dual-target assay was designed for the specific detection of nucleic acid sequences of SARS-CoV-2 in the FAM channel and the Sarbecovirus subgenus family, including SARS-CoV-2, in the HEX channel. SARS-CoV-2 is closely related to SARS-CoV with genomic similarity.

[0106] Sequences were downloaded from NCBI and the Global Initiative on Sharing All Influenza Data (GISAID) database. Seven sequences were downloaded from the GISAID database.

[0107] Sequences from the subgenus Sarbecovirus (taxonomic ID 2509511) were downloaded from the NCBI database. There were 1,094 sequences greater than 200 bases in length. At this time, NCBI classified SARS-CoV-2 as "Wuhan Seafood Market Pneumonia Virus" without assigning a taxonomic ID. To create an inclusion group for SARS-CoV-2, these sarbecovirus sequences were examined to identify viruses labeled "Wuhan Seafood Market Pneumonia Virus." Seven sequences were available and used to identify seven target regions in six non-overlapping regions. The conserved nature of the regions was assessed by comparing them with other sarbecoviruses and then selecting regions within the ORF1a nonstructural region unique to SARS-CoV-2. For pan-sarbecovirus detection, conserved regions within the structural protein envelope E gene and the conserved ORF1a / b region were also selected. The pan-sarbecovirus detection set was also able to detect the novel SARS-CoV-2 virus. This dual-target design strategy replaced single-targeting due to limited sequence availability and the ongoing understanding of the stability of this novel virus. Selective amplification of the RNA internal control was achieved by using non-competitive sequence-specific forward and reverse primers with no homology to the coronavirus genome. Amplification was performed using a thermostable DNA polymerase enzyme.

[0108] The NCBI and GISAID databases were monitored to download newly available sequences and, when available, to obtain taxonomic IDs, countries, and sample collection and sequence deposition dates. These sequences were collected from China, the United States, the United Kingdom, Australia, Japan, Italy, Germany, Finland, France, Nepal, Taiwan, Singapore, and South Korea. There were 175 sequences available between the two databases, and for all but one available viral sequence, the sequences were identical in the SARS-CoV-2 assay target region. MT039890 has a single nucleotide polymorphism (SNP) near the 3' end of the probe hybridization site, which should not affect assay performance.

[0109] Example 3: Selection of SARS-CoV-2 primer and probe oligonucleotides We provided a master mix containing detection probes specific for coronavirus SARS-CoV-2, members of the Sarbecovirus subgenus, and an RNA internal control nucleic acid. The coronavirus and RNA internal control detection probes were each labeled with a unique fluorescent dye that acted as a reporter. Each probe also contained a second dye that acted as a quencher. PCR primers for amplifying the regions of interest were designed to avoid reported SNPS in the target region. Therefore, we initiated testing with a single-well assay design to detect SARS-CoV-2 using: (i) a specific nucleic acid sequence derived from the nonstructural open reading frame (ORF1a / b) of the SARS-CoV-2 genome in one channel (FAM) (nCoV1 assay) and (ii) two whole sarbecovirus assays (pan-1 and / or pan-2) in different channels (HEX) using conserved sequences in the ORF-1 and structural envelope (E) gene locations common to all other sarbecoviruses, including SARS-CoV-2, providing a high degree of robustness. The relative locations of the amplicon targets on the SARS-CoV-2 genome compared to their locations on the SARS-CoV genome are shown in Figure 1. Bioinformatics analysis of SARS-CoV-2 assays that can be multiplexed with the GIC oligonucleotides used for detection of process controls was performed to screen initial assays for performance. Selected combinations of primer sets and probes are shown in Table 5. [Table 5]

[0110] Example 4: PCR assay reagents and conditions Real-time PCR detection of SARS-CoV-2 was performed using the cobas® 6800 / 8800 system platform (Roche Molecular Systems, Inc., Pleasanton, CA). The final concentrations of amplification reagents were as follows: [Table 6] The table below shows a typical thermal profile used in a PCR amplification reaction: [Table 7]

[0111] The pre-PCR program included incubations at 55°C, 60°C, and 65°C for initial denaturation and reverse transcription of the RNA template. The three-temperature incubation has the beneficial effect of suppressing the formation of RNA secondary structures at higher temperatures, thereby resulting in more efficient transcription, while the lower temperatures allow transcription of even slightly mismatched target sequences (e.g., genetic variants of an organism). PCR cycling was divided into two runs, each using a single-stage setup (combined annealing and extension). The first five cycles at 55°C allow for increased inclusiveness by preamplifying slightly mismatched target sequences, while the second run (45 cycles) uses an annealing / extension temperature of 58°C to enhance specificity.

[0112] Example 5: Performance evaluation of SARS-CoV-2 tests Evaluation of components, workflow, and assay reagents for SARS-CoV-2 testing was performed using cobas® 6800 reagents. Linearized recombinant plasmids were tested with assay oligonucleotides to assess performance. In vitro transcripts were also generated to evaluate assay performance using synthetic RNA. Nucleic acid quantification was performed using Qubit with DNA and RNA standards. Plasmid DNA and transcripts were serially diluted in MultiPrep Specimen Diluent Buffer (also known as Bulk Generic Specimen Diluent) and used for assay performance testing. An internal control oligonucleotide (generic internal control, GIC) was included for evaluations using both linearized DNA and RNA transcripts. Experiments were performed on a Roche LC480 cycler equipped and calibrated with a cobas® 6800 filter using the cobas® 6800 generic thermal cycling profile. Nasopharyngeal (NSP) samples were obtained from patients presenting with upper respiratory tract symptoms using agglutination swabs and collected in Universal Viral Transport medium (3 mL). A modified sample preparation workflow (Process and Elution, PnE) was used on the cobas® 6800 System to process either 300 or 400 μL of NSP samples to prepare nucleic acid eluates. These eluates contain gIC sheath RNA (QS RNA control) which serves as an internal sample processing control following the same NSP sample preparation process for the cobas® 6800. The eluates were then used in testing using SARS-CoV-2 assays with amplification and detection on the LC480 and / or cobas® 6800 analytical cyclers.

[0113] Assay oligonucleotides were first evaluated in singleplex assays. Figure 2 shows the performance of the nCoV1 singleplex assay (SEQ ID NOS: 1, 7, and 21) using linearized recombinant plasmids containing target sequences ranging from 1.0E+8 copies (cp) to 1.0E+01 cp per PCR reaction, expressed as amplification curves and mean Ct (n = 3 replicates). The nCoV1 assay demonstrated good sensitivity for detecting up to 10 copies of target sequence per PCR reaction. The assay was also evaluated with in vitro transcripts, and the data are shown in Figure 3. Here, the assay demonstrated good sensitivity for detecting up to 100 copies of target transcript per PCR reaction, with acceptable dynamic range and PCR efficiency, with and without the gIC assay, using synthetic RNA transcripts. Next, the Pan-Sarbeco-2 assay (SEQ ID NOS: 6, 18, and 26) was evaluated using linearized plasmid DNA and transcripts to determine assay sensitivity, and the results are shown in Figure 4.

[0114] Next, a multiplex PCR assay was performed, testing the primer and probe oligonucleotides for the nCoV1 assay (SEQ ID NOs: 1, 7, and 21) and the primer and probe oligonucleotides for the Pan-1 assay (SEQ ID NOs: 5, 15, and 25) in a single reaction. Recombinant plasmids containing the SARS-CoV-2 and sarbecovirus target regions (i.e., ORF1a / b and envelope genes, respectively) were used to generate in vitro transcripts (250 bases and 261 bases). RNA was quantified using a Qubit fluorometer using the RNA standards provided with the assay. To cover a wide dynamic range, serial dilutions of the transcript stock were prepared in sample diluent (SD = Tris buffer) containing carrier RNA at 10x concentration levels of 1e8 to 1e1 copies (cp) / PCR and tested in 10 replicates. PCRs were set up manually using a generic cobas® 6800 / 8800 master mix supplemented with test primers and probes, with amplification and detection in a LightCycler® 480 (LC480) thermocycler fitted with a cobas® 6800 / 8800 filter. Figure 5 shows growth curves and Ct charts (plotting the dynamic range for the two targets) across the levels tested using synthetic in vitro transcripts.

[0115] The data demonstrated robust amplification curves and PCR efficiency over a wide dynamic range, with transcripts detected down to 10 copies per PCR reaction for both targets. Initial limit of detection (LOD) testing data are summarized in Figure 6, which shows a 100% hit rate at levels down to 10 copies / PCR reaction in specimen-diluted samples.

[0116] Example 6: Assay performance of SARS-CoV-2 testing using patient sample isolates Using the Qiagen virus sample preparation protocol, BEI SARS-CoV-2 Isolate USA-WA1 / 2020 (2.8E+5 TCID 50Total genomic viral RNA was isolated from 100% genomic RNA (TCID) of the eluate (100% genomic RNA recovery in the eluate was estimated at 2.8E+5 TCID 50 / mL to 2.80E-02 TCID 50 Ten-fold serial dilutions were prepared up to 7 levels. The final dilution was 20 μL (approximately 5.6 e +3 , 5.6e +2 , 5.6e +1 , 5.6e +0 , 5.6e -1 , 5.6e -2 , 5.6e -3 TCID 50 This was done by adding 5 μL of purified RNA to one of two matrices (10 replicates / mL): a) cobas® Specimen Dilution Buffer / Tris Buffer (SD) at the lower level in 10 replicates and the upper 3 levels in 3 replicates, and b) cobas® 6800 / 8800 System eluate prepared from clinical nasopharyngeal swab specimens from subjects with upper respiratory tract infection symptoms (NSP) in 2 replicates. RNA transcripts in cobas® SD were included as a control.

[0117] Multiplex PCR tests were performed on genomic RNA isolated from patient samples using the primer / probe set for the nCov1 assay (SEQ ID NOs: 1, 7, 21) and the primer / probe set for the whole sarbecovirus-1 assay (SEQ ID NOs: 5, 15, 25). The amplification curves generated in the SD and NSP matrices are shown in Figures 7 and 8, respectively. The Ct values ​​determined from this experiment are summarized in Table 8. [Table 8]

[0118] Testing of SARS-CoV-2 Isolate USA-WA1 / 2020 genomic RNA in specimen diluent yielded a SARS-CoV-2 test of 5.6E -03The results demonstrate that up to TCID50 equivalents of input could be detected (assuming 100% extraction efficiency). Comparing the genomic RNA data with the synthetic transcript copy number data, this corresponds to detection of approximately 10 copies of target template. Furthermore, these results demonstrate that the SARS-CoV-2 test can detect the genomic RNA of isolate USA-WA1 / 2020 in nasopharyngeal matrix (pseudo-NSP system).

[0119] Example 7: Exclusivity / Cross-Reactivity Testing of SARS-CoV-2 Tests The SARS-CoV-2 test was evaluated for exclusivity / cross-reactivity to other respiratory viruses, including MERS and four coronaviruses (229E, OC43, HKU1, and NL63). A list of all viral nucleic acid eluates evaluated is shown in Table 9. No interactions with SARS-CoV-2 were observed, demonstrating the specificity of the test. [Table 9]

[0120] Example 8: SARS-CoV-2 and Influenza A / B Assay Description The SARS-CoV-2 and Influenza A / B test is a multiplex, single-well assay that detects SARS-CoV-2, influenza A, and influenza B viral RNA genome sequences using four distinct channels: the SARS-CoV-2 Dual assay targets (i) specific nucleic acid sequences from the nonstructural open reading frame (ORF1a / b) in the SARS-CoV-2 genome in one channel, and (ii) the conserved structural envelope (E) gene location common to all sarbecoviruses, including SARS-CoV-2, in the second channel. The third channel detects influenza A segment 7 matrix protein 2 (M2) and matrix protein 1 (M1) sequences, and influenza B segment 8 nuclear export protein (NEP) and nonstructural protein 1 (NS1) sequences in the fourth channel. Results demonstrate the specific detection of SARS-CoV-2, influenza A, and influenza B viral RNA genome sequences detectable in nasopharyngeal and oropharyngeal swab samples during the acute phase of infection.

[0121] The SARS-CoV-2 & Influenza A / B test can be performed on the cobas® 6800 / 8800 System, a fully automated system approved / cleared by the FDA for sample preparation (nucleic acid extraction and purification) followed by PCR amplification and detection. Selective amplification of target nucleic acids from samples is achieved through the use of target-specific forward and reverse primers. The master mix contains detection probes specific for SARS-CoV-2, influenza A, and influenza B, as well as an RNA internal control nucleic acid. The SARS-CoV-2, influenza A, influenza B, and RNA internal control detection probes are each labeled with a unique fluorescent dye that acts as a reporter. Amplification of the RNA internal reference is achieved through the use of non-competitive sequence-specific forward and reverse primers that share no homology with the SARS-CoV-2, influenza A, or influenza B genomes. Amplification utilizes a thermostable DNA polymerase enzyme. Each probe also contains a second dye that acts as a quencher. The workflow will use the commercially available cobas® Universal Reagents (MGP cassette, lysis buffer, specimen diluent, and wash buffer) along with the existing reagent cassette containing the proteinase, elution buffer, and MMX R1 (cofactor). The internal control will be used with the newly developed reagents described above. Nucleic acids from patient samples will be simultaneously extracted with a spiked RNA internal control molecule (identical to the existing RNA QS reagent). External controls (positive and negative) will be processed in the same manner for each SARS-CoV-2 and influenza A / B run.

[0122] Example 9: SARS-CoV-2 and Influenza A / B Assay Design Strategy The diagnostic tests are designed for the detection and differentiation of 2019 novel coronavirus (SARS-CoV-2), influenza A, and influenza B. For the SARS-CoV-2 assay, a dual-target assay was designed to detect SARS-CoV-2 in one channel (FAM) and the sarbecovirus subgenus, which also includes SARS-CoV-2, in another channel (HEX). The assay was also designed to detect influenza A sequences in a third channel (COU) and influenza B sequences in a fourth channel (JA270). The SARS-CoV-2 assay contains six oligonucleotides: one reverse transcription (RT) primer for each of two genomic regions, one non-RT primer for each, and one probe labeled with a FAM and HEX fluorophore, respectively. The influenza A assay contains one RT primer, one non-RT primer, and a probe labeled with a COU fluorophore. It detects RNA sequences from influenza A segment 7 matrix protein 2 (M2) and matrix protein 1 (M1). The influenza B assay is also designed as a Pan-FluB assay to detect all common influenza B strains, including two influenza B lineages: B / Yamagata and B / Victoria. The assay contains one RT primer, one non-RT primer, and a probe labeled with the JA270 fluorophore. It detects influenza B segment 8 nuclear export protein (NEP) and nonstructural protein 1 (NS1) sequences.

[0123] Example 10: SARS-CoV-2 and Influenza A / B Assay Primer and Probe Selection The master mix contains detection probes specific for coronavirus type SARS-CoV-2, members of the Sarbecovirus subgenus, and for influenza A and B, as well as RNA internal control nucleic acids, each with its own fluorophore. The relative locations of the amplicon targets on the SARS-CoV-2, influenza A, and influenza B genomes are shown in Figures 9A, 9B, and 9C, respectively. Selected combinations of primer sets and probes are shown in Table 10. [Table 10]

[0124] Example 10: Performance evaluation of SARS-CoV-2 and influenza A / B tests The feasibility evaluation of the components (including selected combinations of primers and probes shown in Table 10), workflow, and assay reagents for the combined SARS-CoV-2 & Influenza A / B test is based on experimental evaluation from a study using cobas® 6800 reagents. Linearized recombinant plasmids were tested with the assay oligonucleotides to evaluate performance. In vitro transcripts were also generated to evaluate assay performance using synthetic RNA. Nucleic acid quantification was performed using Qubit with DNA and RNA standards. Plasmid DNA and transcripts were serially diluted in MultiPrep Specimen Diluent Buffer (also known as BGSD, MPSD) and used for assay performance testing. An internal control oligonucleotide (universal internal control, GIC) was included for evaluations using both linearized DNA and RNA transcripts. Experiments were performed on a Roche Z480 equipped with a 6800 filter cycler fitted and calibrated with a cobas® 6800 filter, using the cobas® 6800 universal thermal cycling profile described in Example 4.

[0125] Nasopharyngeal (NPS) samples were obtained from patients presenting with upper respiratory tract symptoms using agglutination swabs and collected in Universal Viral Transport medium (3 mL). These samples were characterized by an in-house developed PCR assay and shown to exclude the following viruses: FluA, FluB, RSV, HMPV, AdV, EV / RV, and HPIV1, HPIV2, HPIV3, HPIV4, and human coronaviruses (229E, NL63, HKU1, and OC43) by MiSeq sequencing of the PCR products. These NPS samples were stored frozen at -70°C, thawed, and used for experiments as needed. A modified sample preparation workflow (process and eluate, P&E) was used on the cobas® 6800 System to process either 300 or 400 μL of NPS samples to prepare nucleic acid eluates. These eluates contain gIC sheath RNA (QS RNA control), which serves as an internal sample processing control, following the same NPS sample preparation process for the cobas® 6800 System. The eluate was then used in testing using a SARS-CoV-2 assay with amplification and detection on a Z480 equipped with a 6800 filter cycler and / or a cobas® 6800 analytical cycler.

[0126] Example 11: Linearity and limits of detection with transcripts in a clean system Experiments were first performed with synthetic transcripts corresponding to each target assay region using the clean system (BGSD) with the primer sets and probe combinations listed in Table 10. Four transcripts were pooled at high copy levels and tested at several dilution levels (more replicates at lower levels) in a 6800-μL PCR system. The results in Figures 10, 11, 12, and Table 11 show good reproducibility at high input levels for the pan-sarbecovirus and FluB assays, low standard deviation at lower input levels, and only one dropout at 5 cp / PCR, respectively. Excellent sensitivity of each assay was demonstrated across a wide dynamic range (1E+9 to 1E+1 cp) in PCR. Good linearity and high PCR efficiencies ranging from 106% to 112% were observed for SARS-CoV-2, influenza A, and influenza B targets. [Table 11]

[0127] Example 12: Linearity using virus cultures in simulated nasopharyngeal matrices Three genomic RNA eluates from commercial virus cultures were pooled at high copy levels and tested on the cobas® 6800 system at several dilution levels (n=2 / level). Results in Table 12 and Figure 13 show good performance across an 8-log dynamic range, with all levels producing good detectable signals. Using droplet digital PCR copy number estimates of TCID50 for each virus lot, low-end detection per PCR is calculated to be <2 copies, <10 copies, and <30 copies for influenza A, SARS-CoV-2, and influenza B, respectively. [Table 12]

[0128] Although the foregoing invention has been described in some detail for purposes of clarity and understanding, it will be apparent to those skilled in the art upon reading this disclosure that various changes in form and detail can be made. For example, all of the techniques and devices described above can be used in various combinations.

Claims

1. 1. A method for detecting severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in a sample, comprising: - performing an amplification step, which comprises contacting the sample with a primer set to produce an amplification product if SARS-CoV-2 nucleic acid is present in the sample; - carrying out a hybridization step comprising contacting said amplification products with one or more detectable probes; - detecting the presence or absence of said amplification product, wherein the presence of said amplification product indicates the presence of SARS-CoV-2 in said sample and the absence of said amplification product indicates the absence of SARS-CoV-2 in said sample; Including, the first primer comprises the oligonucleotide sequence of SEQ ID NO: 1 or 27-29; the second primer comprises the oligonucleotide sequence of SEQ ID NO: 7; and the detectable probe comprises the oligonucleotide sequence of SEQ ID NO: 21 or 42, or a complement thereof; or The method, wherein the first primer comprises the oligonucleotide sequence of SEQ ID NO: 40; the second primer comprises the oligonucleotide sequence of SEQ ID NO: 41; and the detectable probe comprises the oligonucleotide sequence of SEQ ID NO: 43, or a complement thereof.

2. the hybridizing step comprises contacting the amplification product with the detectable probe labeled with a donor fluorescent moiety and a corresponding acceptor moiety; 10. The method of claim 1, wherein the detecting step comprises detecting the presence or absence of fluorescence resonance energy transfer (FRET) between the donor fluorescent moiety and the acceptor moiety of the probe, wherein the presence or absence of fluorescence indicates the presence or absence of SARS-CoV-2 in the sample.

3. 3. The method of claim 2, wherein the amplification step uses a polymerase enzyme with 5'-3' nuclease activity.

4. conducting an amplification step in the presence of a second primer set comprising a first primer comprising an oligonucleotide sequence selected from the group consisting of SEQ ID NOs: 4-6 and a second primer comprising an oligonucleotide sequence selected from the group consisting of SEQ ID NOs: 15-20 to generate a second amplification product if SARS-CoV-2 or SARS-CoV-2 and other coronavirus target nucleic acids from the subgenus Sarbecovirus are present in the sample; and 4. The method of any one of claims 1 to 3, comprising carrying out a hybridizing step in the presence of a detectable probe comprising an oligonucleotide sequence selected from the group consisting of SEQ ID NOs: 24-26 and 32, or a complement thereof, wherein detecting the presence of a second amplification product indicates the presence of SARS-CoV-2 or other coronavirus target nucleic acid from SARS-CoV-2 and the subgenus Sarbecovirus in the sample, and wherein the absence of an amplification product indicates the absence of SARS-CoV-2 or other coronavirus target nucleic acid from SARS-CoV-2 and the subgenus Sarbecovirus in the sample.

5. the primer set for amplifying SARS-CoV-2 comprises a plurality of first primers, a plurality of second primers, and a plurality of detectable probes; the plurality of first primers are a combination of a first primer comprising an oligonucleotide sequence selected from the group consisting of SEQ ID NOs: 1 and 27 and a first primer comprising the oligonucleotide sequence of SEQ ID NO: 5; the plurality of second primers are a combination of a second primer comprising the oligonucleotide sequence of SEQ ID NO: 7 and a second primer comprising the oligonucleotide sequence of SEQ ID NO: 15; 5. The method of any one of claims 1 to 4, wherein the plurality of detectable probes is a combination of an oligonucleotide probe comprising the oligonucleotide sequence of SEQ ID NO: 21 or 42, or a complement thereof, and an oligonucleotide probe comprising an oligonucleotide sequence selected from the group consisting of SEQ ID NO: 25 and 32, or a complement thereof.

6. 6. The method of any one of claims 1 to 5, further comprising detecting nucleic acids from one or more other viruses in parallel, wherein the one or more other viruses are selected from the group consisting of influenza virus, bat coronavirus, severe acute respiratory syndrome (SARS) coronavirus (SARS-CoV) and Middle East respiratory syndrome (MERS) coronavirus (MERS-CoV), coronavirus (229E, NL63, OC43, HKU1), respiratory syncytial virus, human metapneumovirus, adenovirus (B, E, U, C), enterovirus, rhinovirus, and human parainfluenza virus (1, 2, 3, 4), and any combination thereof.

7. 10. The method of claim 6, further comprising: performing an amplification step in the presence of an influenza A primer set for generating an amplification product if influenza A is present in the sample; and an influenza B primer set for generating an amplification product if influenza B is present in the sample; and performing a hybridization step in the presence of one or more detectable influenza A probes and one or more influenza B probes, wherein detecting the presence of an influenza A amplification product indicates the presence of influenza A in the sample, and the absence of an influenza A amplification product indicates the absence of influenza A in the sample, and detecting the presence of an influenza B amplification product indicates the presence of influenza B in the sample, and the absence of an influenza B amplification product indicates the absence of influenza B in the sample.

8. the set of influenza A primers comprises a forward primer comprising the oligonucleotide sequence of SEQ ID NO: 33 and a reverse primer comprising the oligonucleotide sequence of SEQ ID NO: 34; and one of the one or more detectable influenza A probes comprises the oligonucleotide sequence of SEQ ID NO: 35 or 44, or a complement thereof; and / or 8. The method of claim 7, wherein the set of influenza B primers comprises a forward primer comprising the oligonucleotide sequence of SEQ ID NO: 36 and a reverse primer comprising the oligonucleotide sequence of SEQ ID NO: 37; and one of the one or more detectable influenza B probes comprises the oligonucleotide sequence of SEQ ID NO: 38 or 45, or a complement thereof.

9. A kit for detecting SARS-CoV-2 nucleic acid, comprising: a first primer comprising a first oligonucleotide sequence selected from the group consisting of SEQ ID NOs: 1 and 27 to 29; a second primer comprising a second oligonucleotide sequence of SEQ ID NO: 7; a third fluorescently detectably labeled probe comprising an oligonucleotide sequence selected from the group consisting of SEQ ID NOs: 21 and 42, or a complement thereof, wherein the third fluorescently detectably labeled probe is configured to hybridize to the amplicon generated by the first primer and the second primer; or a first primer comprising a first oligonucleotide sequence of SEQ ID NO: 40; a second primer comprising a second oligonucleotide sequence of SEQ ID NO: 41; a third fluorescently detectably labeled probe comprising the oligonucleotide sequence of SEQ ID NO: 43, or a complement thereof, wherein the third fluorescently detectably labeled probe is configured to hybridize to the amplicon generated by the first primer and the second primer; Includes a kit.

10. 10. The kit of claim 9, wherein the third detectably labeled probe comprises a donor fluorescent moiety and a corresponding acceptor moiety.

11. 11. The kit of claim 9 or 10, further comprising nucleoside triphosphates, a nucleic acid polymerase, and a buffer necessary for the function of the nucleic acid polymerase.

12. 12. The kit of any one of claims 9 to 11, wherein at least one of the first primer, the second primer, and the third fluorescently detectably labeled probe comprises at least one modified nucleotide.

13. 13. The kit of any one of claims 9 to 12, further comprising: a first primer comprising an oligonucleotide sequence selected from the group consisting of SEQ ID NOs: 4 to 6; a second primer comprising an oligonucleotide sequence selected from the group consisting of SEQ ID NOs: 15 to 20; and a detectable probe comprising an oligonucleotide sequence selected from the group consisting of SEQ ID NOs: 24 to 26, and 32, or a complement thereof.

14. 14. The kit of claim 13, wherein the first primer comprises the oligonucleotide sequence of SEQ ID NO: 5, the second primer comprises the oligonucleotide sequence of SEQ ID NO: 15, and the detectable probe comprises the oligonucleotide sequence of SEQ ID NO: 25 or 32 or a complement thereof.

15. 15. The kit of any one of claims 9 to 14, wherein the kit is for simultaneously detecting severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), influenza A, and influenza B, and further comprises a set of influenza A primers and one or more detectable influenza A probes, and a set of influenza B primers and one or more detectable influenza B probes.

16. the set of influenza A primers comprises a forward primer comprising the oligonucleotide sequence of SEQ ID NO: 33 and a reverse primer comprising the oligonucleotide sequence of SEQ ID NO: 34; and one of the one or more detectable influenza A probes comprises the oligonucleotide sequence of SEQ ID NO: 35 or 44; and / or 16. The kit of claim 15, wherein the set of influenza B primers comprises a forward primer comprising the oligonucleotide sequence of SEQ ID NO: 36 and a reverse primer comprising the oligonucleotide sequence of SEQ ID NO: 37; and one of the one or more detectable influenza B probes comprises the oligonucleotide sequence of SEQ ID NO: 38 or 45.

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