WO2022079224A1

WO2022079224A1 - Methods and kits for detecting sars-cov-2

Info

Publication number: WO2022079224A1
Application number: PCT/EP2021/078576
Authority: WO
Inventors: Pascal Pineau; Agnès Marchio
Original assignee: Institut Pasteur
Priority date: 2020-10-15
Filing date: 2021-10-15
Publication date: 2022-04-21

Abstract

In vitro methods for detecting SARS-CoV-2 in a sample are provided. In some embodiments the methods comprise characterizing the replicative activity of SARS-CoV- 2 in a sample. Also provided are methods for diagnosing whether a patient who is a SARS-CoV-2 carrier has COVID-19 or is at an elevated risk of developing COVID-19; methods for characterizing a COVID-19 treatment; methods for determining whether a patient who is a SARS-CoV-2 carrier is contagious. Kits for performing the methods are also provided.

Description

METHODS AND KITS FOR DETECTING SARS-COV-2

BACKGROUND

[0001] Seventeen years after the epidemics of Severe Acute Respiratory Syndrome (SARS) that started from Foshan, in the south China province of Guangdong, another far more contagious coronaviral zoonosis emerged in December 2019 in the city of Wuhan, capital of the Hubei province (Central China) (1 , 2). This novel disease, due to a previously unknown member of the Coronaviridae family was called SARS-CoV-2 after the initial SARS-CoV-1 with whom it shares 82% nucleotide identity (3, 4). The disease itself was named COronaVirus Disease 2019 (COVID-19) by the World Health Organization on February 11 , 2020.

[0002] The diagnosis of the disease is commonly based on the amplification by reverse- transcribed quantitative PCR (RT-qPCR) of at least two different fragments of SARS-CoV-2 RNA genome (5). At the end of the disease, patients are considered to be cured if asymptomatic and when two PCR diagnoses on respiratory samples taken 24h apart are negative (6). Territories and timing of sampling are paramount for diagnosis of COVID-19 (7). The replication of SARS-CoV-2 is characterized by an apparent downward migration from epithelia in the nasal cavity to that of the throat, and then to pulmonary alveoli. As a consequence, viral genome copy numbers tend to be higher in the naso-pharyngeal compartment at the onset of symptoms, then to decline progressively (8, 9). In severe forms of COVID-19, where patients are more extensively explored, the viral genome is more easily detected in lower respiratory tract samples such as broncho-alveolar lavage fluid (BALF) or sputum than in extracts of specimens from upper respiratory locations (9). Infection territories extend, however, well beyond respiratory tract and although SARS-Cov-2 genomes are infrequently detected in patient blood samples, a protracted viral shedding in feces is found by rectal swabs analysis in a significant proportion of patients (10, 11 ). [0003] Nevertheless, COVID-19 diagnosis is considered to be unfortunately characterized by a significant number of false negative results (12, 13). This situation has been emphasized from the onset of the pandemics by radiologists using thoracic CT scan who claimed to be more sensitive than quantitative real-time PCR detection. Different issues are presumably responsible for theses failures. First, easily accessible upper respiratory tract is frequently not anymore proficient for viral replication at patient presentation to clinicians. Second, for unknown reasons, viral replication appears as massively fluctuating in a given organ as observed in most initial clinical descriptions of COVID-19. As a consequence, in a significant proportion of cases, the sequence of sampling outcomes in a given patient is marked by the succession of positive and negative results for SARS-CoV-2 RNA presence (14, 15).

[0004] The improvement of virus detection in COVID-19 remains a priority in clinical practice both to diagnose new cases and to authorize the discharge of the patients. It represents also a desirable evolution to better understand virus circulation between index cases and their contacts, to characterize asymptomatic or paucisymptomatic patients, or to detect the infectious agent in samples such as blood and urine deemed to be poor in SARS-CoV-2 RNA. Several technical options including loop-mediated isothermal amplification (LAMP) coupled or not with CRISPR-Cas technology, have been recently developed and represent potentially promising evolutions of COVID-19 diagnosis (16, 17). We accumulated in recent years some experience in the detection by droplet digital PCR (ddPCR) of low biotic burden (somatic mutants or viruses) in the blood of patients with liver cancer (18, 19). There is a need for new PCR-based methods and reagents to detect SARS-CoV-2, to diagnose COVID19, and for many other uses. This invention meets these and other needs.

SUMMARY OF THE INVENTION

[0005] The examples present data demonstrating the use of new nonobvious methods of detecting SARS-CoV-2 RNA. Based in part on this data, this application provides several new and nonobvious products and methods. [0006] In vitro methods for detecting SARS-CoV-2 in a sample

[0007] In a first aspect the invention provides in vitro methods for detecting SARS- CoV-2 in a sample. The methods comprise providing at least one subject sample; subjecting the at least one sample to a reverse transcription reaction to generate a cDNA copy of RNA in the at least one sample; amplifying any resultant cDNA formed using a first forward primer and a first reverse primer that amplify all or part of a first SARS-CoV- 2 gene selected from the group consisting of spike (S), envelope (E), membrane (M), nucleocapsid (N), accessory protein (ap) 3a (ap3a), ap3b, ap3c, ap3d, ap6, ap7a, ap7b, ap8, ap9a, ap9b, ap9c, ap10 and ap14, to form a first amplified product; detecting any first amplified product formed using a first probe that hybridizes to the first amplified product; amplifying any resultant cDNA formed using a second forward primer and a second reverse primer that amplify all or part of a second SARS-CoV-2 gene selected from the group consisting of non-structural protein (nsp) 1 (nsp1 ), nsp1 , nsp2, nsp3, nsp4, nsp5, nsp6, nsp7, nsp8, nsp9, nsp10, nsp11 , nsp12, nsp13, nsp14, nsp15 and nsp16, to form a second amplified product; and detecting any second amplified product formed using a second probe that hybridizes to the second amplified product. In some embodiments the first SARS-CoV-2 gene is selected from the group consisting of N, E, and ap7a. In some embodiments the second SARS-CoV-2 gene is selected from the group consisting of nsp12 and nsp13.

[0008] In some embodiments of the methods:

[0009] (A) when the first SARS-CoV-2 gene is N the first forward primer hybridizes to the sequence

and the RNA equivalent thereof; the first reverse primer hybridizes to the sequence

(SEQ ID NO: 15) and the RNA equivalent thereof; and the first probe hybridizes to the sequence and the RNA equivalent

thereof;

[0010] (B) when the first SARS-CoV-2 gene is E the first forward primer hybridizes to the sequence and the RNA

equivalent thereof; the first reverse primer hybridizes to the sequence TGTGTGCGTACTGCTGCAATAT (SEQ ID NO: 21 ) and the RNA equivalent thereof; and the first probe hybridizes to the sequence CGAAGCGCAGTAAGGATGGCTAGTGT (SEQ ID NO: 22) and the RNA equivalent thereof;

[0011] (C) when the second SARS-CoV-2 gene is nsp12 the second forward primer hybridizes to the sequence CGAAATCATACCAGTTACC (SEQ ID NO: 17) and the RNA equivalent thereof; the second reverse primer hybridizes to the sequence CCTATATTAACCTTGACCAG (SEQ ID NO: 18) and the RNA equivalent thereof; and the second probe hybridizes to the sequence CCTGGCGTGGTTTGTATGA (SEQ ID NO: 19) and the RNA equivalent thereof; and

[0012] (D) when the second SARS-CoV-2 gene is nsp13 the second forward primer hybridizes to the sequence CTGGTTCTAGTGTGCCCTTA (SEQ ID NO: 23) and the RNA equivalent thereof; the second reverse primer hybridizes to the sequence CGTTGTCCTGCTGAAATTGT (SEQ ID NO: 24) and the RNA equivalent thereof; and the second probe hybridizes to the sequence TTCCGAGGAACATGTCTGGACCT (SEQ ID NO: 25) and the RNA equivalent thereof.

[0013] In some embodiments of the methods:

[0014] (A) when the first SARS-CoV-2 gene is N the first forward primer comprises the sequence GGGGAACTTCTCCTGCTA (SEQ ID NO: 2) and the RNA equivalent thereof; the first reverse primer comprises the sequence CAGACATTTTGCTCTCAA (SEQ ID NO: 3) and the RNA equivalent thereof; and the first probe comprises the sequence TTGCTGCTGCTTGACAGATT (SEQ ID NO: 4) and the RNA equivalent thereof;

[0015] (B) when the first SARS-CoV-2 gene is E the first forward primer comprises the sequence ACAGGTACGTTAATAGTTAATAGCGT (SEQ ID NO: 8) and the RNA equivalent thereof; the first reverse primer comprises the sequence ATATTGCAGCAGTACGCACACA (SEQ ID NO: 9) and the RNA equivalent thereof; and the first probe comprises the sequence ACACTAGCCATCCTTACTGCGCTTCG (SEQ ID NO: 10) and the RNA equivalent thereof;

[0016] (C) when the second SARS-CoV-2 gene is nsp12 the second forward primer comprises the sequence GGTAACTGGTATGATTTCG (SEQ ID NO: 5) and the RNA equivalent thereof; the second reverse primer comprises the sequence CTGGTCAAGGTTAATATAGG (SEQ ID NO: 6) and the RNA equivalent thereof; and the second probe comprises the sequence TCATACAAACCACGCCAGG (SEQ ID NO: 7) and the RNA equivalent thereof; and

[0017] (D) when the second SARS-CoV-2 gene is nsp13 the second forward primer comprises the sequence TAAGGGCACACTAGAACCAG (SEQ ID NO: 1 1 ) and the RNA equivalent thereof; the second reverse primer comprises the sequence ACAATTTCAGCAGGACAACG (SEQ ID NO: 12) and the RNA equivalent thereof; and the second probe comprises the sequence AGGTCCAGACATGTTCCTCGGAA (SEQ ID NO: 13) and the RNA equivalent thereof.

[0018] In some embodiments of the methods:

[0019] (A) when the first SARS-CoV-2 gene is N the first forward primer consists of the sequence GGGGAACTTCTCCTGCTA (SEQ ID NO: 2) and the RNA equivalent thereof; the first reverse primer consists of the sequence CAGACATTTTGCTCTCAA (SEQ ID NO: 3) and the RNA equivalent thereof; and the first probe consists of the sequence TTGCTGCTGCTTGACAGATT (SEQ ID NO: 4) and the RNA equivalent thereof;

[0020] (B) when the first SARS-CoV-2 gene is E the first forward primer consists of the sequence ACAGGTACGTTAATAGTTAATAGCGT (SEQ ID NO: 8) and the RNA equivalent thereof; the first reverse primer consists of the sequence ATATTGCAGCAGTACGCACACA (SEQ ID NO: 9) and the RNA equivalent thereof; and the first probe consists of the sequence ACACTAGCCATCCTTACTGCGCTTCG (SEQ ID NO: 10) and the RNA equivalent thereof; [0021] (C) when the second SARS-CoV-2 gene is nsp12 the second forward primer consists of the sequence GGTAACTGGTATGATTTCG (SEQ ID NO: 5) and the RNA equivalent thereof; the second reverse primer consists of the sequence CTGGTCAAGGTTAATATAGG (SEQ ID NO: 6) and the RNA equivalent thereof; and the second probe consists of the sequence TCATACAAACCACGCCAGG (SEQ ID NO: 7) and the RNA equivalent thereof; and

[0022] (D) when the second SARS-CoV-2 gene is nsp13 the second forward primer consists of the sequence TAAGGGCACACTAGAACCAG (SEQ ID NO: 11 ) and the RNA equivalent thereof; the second reverse primer consists of the sequence ACAATTTCAGCAGGACAACG (SEQ ID NO: 12) and the RNA equivalent thereof; and the second probe consists of the sequence AGGTCCAGACATGTTCCTCGGAA (SEQ ID NO: 13) and the RNA equivalent thereof.

[0023] In some embodiments of the methods amplification of the first amplified product using the first forward primer and the first reverse primer is performed in a first container and amplification of the second amplified product using the second forward primer and the second reverse primer is performed in a second container.

[0024] In some embodiments of the methods amplification of the first amplified product using the first forward primer and the first reverse primer, and amplification of the second amplified product using the second forward primer and the second reverse primer are performed in the same container.

[0025] In some embodiments the methods further comprise quantifying the RNA transcripts of the first SARS-CoV-2 gene in the at least one sample and/or quantifying the RNA transcripts of the second SARS-CoV-2 gene in the at least one sample.

[0026] In some embodiments the methods further comprise calculating the ratio of the quantified RNA transcripts of the first SARS-CoV-2 gene to the quantified RNA transcripts of the second SARS-CoV-2 gene and comparing the calculated ratio to a threshold value; wherein, when the value of the ratio is higher than a threshold value, the SARS-CoV-2 is replicating in the sample.

[0027] In some embodiments of the methods the amplification(s) are performed using a real-time PCR (RT-PCR) or quantitative PCR (qPCR) method.

[0028] In some embodiments of the methods the amplification(s) are performed using a droplet digital PCR (ddPCR) method.

[0029] In vitro methods for characterizing the replicative activity of SARS-CoV-2 in a sample

[0030] In another aspect this invention provides in vitro methods for characterizing the replicative activity of SARS-CoV-2 in a sample. The methods comprise providing at least one subject sample; subjecting the at least one sample to a reverse transcription reaction to generate a cDNA copy of RNA in the at least one sample; amplifying any resultant cDNA formed using a first forward primer and a first reverse primer that amplify all or part of a first SARS-CoV-2 gene selected from the group consisting of spike (S), envelope (E), membrane (M), nucleocapsid (N), accessory protein (ap) 3a (ap3a), ap3b, ap3c, ap3d, ap6, ap7a, ap7b, ap8, ap9a, ap9b, ap9c, ap10 and ap14, to form a first amplified product; detecting any first amplified product formed using a first probe that hybridizes to the first amplified product; amplifying any resultant cDNA formed using a second forward primer and a second reverse primer that amplify all or part of a second SARS-CoV-2 gene selected from the group consisting of non-structural protein (nsp) 1 (nsp1 ), nsp1 , nsp2, nsp3, nsp4, nsp5, nsp6, nsp7, nsp8, nsp9, nsp10, nsp1 1 , nsp12, nsp13, nsp14, nsp15 and nsp16, to form a second amplified product; detecting any second amplified product formed using a second probe that hybridizes to the second amplified product; quantifying the RNA transcripts of the first SARS-CoV-2 gene in the at least one sample and quantifying the RNA transcripts of the second SARS-CoV-2 gene in the at least one sample; and calculating the ratio of the quantified RNA transcripts of the first SARS-CoV-2 gene to the quantified RNA transcripts of the second SARS-CoV-2 gene and comparing the calculated ratio to a threshold value; wherein, when the value of the ratio is higher than a threshold value, the SARS-CoV-2 is replicating in the sample. In some embodiments the first SARS-CoV-2 gene is selected from the group consisting of N, E, and ap7a. In some embodiments the second SARS- CoV-2 gene is selected from the group consisting of nsp12 and nsp13.

[0031] In some embodiments of the methods:

[0032] (A) when the first SARS-CoV-2 gene is N the first forward primer hybridizes to the sequence TAGCAGGAGAAGTTCCCC (SEQ ID NO: 14) and the RNA equivalent thereof; the first reverse primer hybridizes to the sequence TTGAGAGCAAAATGTCTG (SEQ ID NO: 15) and the RNA equivalent thereof; and the first probe hybridizes to the sequence AATCTGTCAAGCAGCAGCAA (SEQ ID NO: 16) and the RNA equivalent thereof;

[0033] (B) when the first SARS-CoV-2 gene is E the first forward primer hybridizes to the sequence ACGCTATTAACTATTAACGTACCTGA (SEQ ID NO: 20) and the RNA equivalent thereof; the first reverse primer hybridizes to the sequence TGTGTGCGTACTGCTGCAATAT (SEQ ID NO: 21 ) and the RNA equivalent thereof; and the first probe hybridizes to the sequence CGAAGCGCAGTAAGGATGGCTAGTGT (SEQ ID NO: 22) and the RNA equivalent thereof;

[0034] (C) when the second SARS-CoV-2 gene is nsp12 the second forward primer hybridizes to the sequence CGAAATCATACCAGTTACC (SEQ ID NO: 17) and the RNA equivalent thereof; the second reverse primer hybridizes to the sequence CCTATATTAACCTTGACCAG (SEQ ID NO: 18) and the RNA equivalent thereof; and the second probe hybridizes to the sequence CCTGGCGTGGTTTGTATGA (SEQ ID NO: 19) and the RNA equivalent thereof; and

[0035] (D) when the second SARS-CoV-2 gene is nsp13 the second forward primer hybridizes to the sequence CTGGTTCTAGTGTGCCCTTA (SEQ ID NO: 23) and the RNA equivalent thereof; the second reverse primer hybridizes to the sequence CGTTGTCCTGCTGAAATTGT (SEQ ID NO: 24) and the RNA equivalent thereof; and the second probe hybridizes to the sequence TTCCGAGGAACATGTCTGGACCT (SEQ ID NO: 25) and the RNA equivalent thereof.

[0036] In some embodiments of the methods:

[0037] (A) when the first SARS-CoV-2 gene is N the first forward primer comprises the sequence GGGGAACTTCTCCTGCTA (SEQ ID NO: 2) and the RNA equivalent thereof; the first reverse primer comprises the sequence CAGACATTTTGCTCTCAA (SEQ ID NO: 3) and the RNA equivalent thereof; and the first probe comprises the sequence TTGCTGCTGCTTGACAGATT (SEQ ID NO: 4) and the RNA equivalent thereof;

[0038] (B) when the first SARS-CoV-2 gene is E the first forward primer comprises the sequence ACAGGTACGTTAATAGTTAATAGCGT (SEQ ID NO: 8) and the RNA equivalent thereof; the first reverse primer comprises the sequence ATATTGCAGCAGTACGCACACA (SEQ ID NO: 9) and the RNA equivalent thereof; and the first probe comprises the sequence ACACTAGCCATCCTTACTGCGCTTCG (SEQ ID NO: 10) and the RNA equivalent thereof;

[0039] (C) when the second SARS-CoV-2 gene is nsp12 the second forward primer comprises the sequence GGTAACTGGTATGATTTCG (SEQ ID NO: 5) and the RNA equivalent thereof; the second reverse primer comprises the sequence CTGGTCAAGGTTAATATAGG (SEQ ID NO: 6) and the RNA equivalent thereof; and the second probe comprises the sequence TCATACAAACCACGCCAGG (SEQ ID NO: 7) and the RNA equivalent thereof; and

[0040] (D) when the second SARS-CoV-2 gene is nsp13 the second forward primer comprises the sequence TAAGGGCACACTAGAACCAG (SEQ ID NO: 1 1 ) and the RNA equivalent thereof; the second reverse primer comprises the sequence ACAATTTCAGCAGGACAACG (SEQ ID NO: 12) and the RNA equivalent thereof; and the second probe comprises the sequence AGGTCCAGACATGTTCCTCGGAA (SEQ ID NO: 13) and the RNA equivalent thereof. [0041] In some embodiments of the methods:

[0042] (A) when the first SARS-CoV-2 gene is N the first forward primer consists of the sequence GGGGAACTTCTCCTGCTA (SEQ ID NO: 2) and the RNA equivalent thereof; the first reverse primer consists of the sequence CAGACATTTTGCTCTCAA (SEQ ID NO: 3) and the RNA equivalent thereof; and the first probe consists of the sequence TTGCTGCTGCTTGACAGATT (SEQ ID NO: 4) and the RNA equivalent thereof;

[0043] (B) when the first SARS-CoV-2 gene is E the first forward primer consists of the sequence ACAGGTACGTTAATAGTTAATAGCGT (SEQ ID NO: 8) and the RNA equivalent thereof; the first reverse primer consists of the sequence ATATTGCAGCAGTACGCACACA (SEQ ID NO: 9) and the RNA equivalent thereof; and the first probe consists of the sequence ACACTAGCCATCCTTACTGCGCTTCG (SEQ ID NO: 10) and the RNA equivalent thereof;

[0044] (C) when the second SARS-CoV-2 gene is nsp12 the second forward primer consists of the sequence GGTAACTGGTATGATTTCG (SEQ ID NO: 5) and the RNA equivalent thereof; the second reverse primer consists of the sequence CTGGTCAAGGTTAATATAGG (SEQ ID NO: 6) and the RNA equivalent thereof; and the second probe consists of the sequence TCATACAAACCACGCCAGG (SEQ ID NO: 7) and the RNA equivalent thereof; and

[0045] (D) when the second SARS-CoV-2 gene is nsp13 the second forward primer consists of the sequence TAAGGGCACACTAGAACCAG (SEQ ID NO: 11 ) and the RNA equivalent thereof; the second reverse primer consists of the sequence ACAATTTCAGCAGGACAACG (SEQ ID NO: 12) and the RNA equivalent thereof; and the second probe consists of the sequence AGGTCCAGACATGTTCCTCGGAA (SEQ ID NO: 13) and the RNA equivalent thereof.

[0046] In some embodiments of the methods amplification of the first amplified product using the first forward primer and the first reverse primer is performed in a first container and amplification of the second amplified product using the second forward primer and the second reverse primer is performed in a second container.

[0047] In some embodiments of the methods amplification of the first amplified product using the first forward primer and the first reverse primer, and amplification of the second amplified product using the second forward primer and the second reverse primer are performed in the same container.

[0048] Other Methods

[0049] The methods for characterizing the replicative activity of SARS-CoV-2 in a sample may be used in additional methods of characterization, diagnosis and treatment.

[0050] Therefore, this invention also provides an in vitro method for diagnosing whether a patient who is a SARS-CoV-2 carrier has COVID-19 or is at an elevated risk of developing COVID-19. The method includes characterizing the replicative activity of SARS-CoV-2 in a sample by a method described above, to determine whether SARS- CoV-2 is replicating in the sample. If SARS-CoV-2 is replicating in the sample the patient is diagnosed with COVID-19 or with an elevated risk of developing COVID-19; and/or if SARS-CoV-2 is not replicating in the sample the patient is not diagnosed with COVID- 19 or with an elevated risk of developing COVID-19.

[0051] The invention also provides an in vitro method for characterizing a SARS- CoV-2 infection in a patient over time. The method includes taking a first sample from a patient at a first timepoint; characterizing the replicative activity of SARS-CoV-2 in the first sample by a method described above to determine whether SARS-CoV-2 is replicating in the first sample at the first timepoint; taking a second sample from the patient at a second timepoint; and characterizing the replicative activity of SARS-CoV-2 in the second sample by a method described above to determine whether SARS-CoV-2 is replicating in the second sample at the second timepoint. In some embodiments replication of SARS-CoV-2 is higher in the second sample than the first sample and the COVID-19 status of the patient is worsening. In some embodiments replication of SARS- CoV-2 is lower in the second sample than the first sample and the COVID-19 status of the patient is improving. In some embodiments the method further includes administering a COVID-19 treatment to the patient after the first timepoint and before the second timepoint.

[0052] This invention also provides an in vitro method for characterizing a COVID- 19 treatment. The method comprises administering a COVID-19 treatment to a patient diagnosed as a SARS-CoV-2 carrier and/or diagnosed with COVID-19 and taking a sample from the patient. The method includes characterizing the replicative activity of SARS-CoV-2 in a sample by a method described above, to determine whether SARS- CoV-2 is replicating in the sample. If SARS-CoV-2 is replicating in the sample the treatment is determined to not be effective; and/or if SARS-CoV-2 is not replicating in the sample the treatment is determined to be effective. In some embodiments the treatment is a COVID-19 vaccine. In some embodiments the treatment is not a COVID- 19 vaccine. In some embodiments the treatment is a non-vaccine agent that reduces or eliminates at least one symptom of COVID-19.

[0053] This invention also provides an in vitro method for determining whether a patient who is a SARS-CoV-2 carrier is contagious. The method includes characterizing the replicative activity of SARS-CoV-2 in a sample by a method described above, to determine whether SARS-CoV-2 is replicating in the sample. If SARS-CoV-2 is replicating in the sample the patient is diagnosed as contagious; and/or if SARS-CoV-2 is not replicating in the sample the patient is not diagnosed as contagious.

[0054] Kits

[0055] The invention also provides kits for detecting SARS-CoV-2 in a sample.

[0056] In some embodiments the kits may be used to characterize the replicative activity of SARS-CoV-2 in a sample. [0057] In some embodiments the kits may be used in an in vitro method for diagnosing whether a patient who is a SARS-CoV-2 carrier has COVID-19 or is at an elevated risk of developing COVID-19.

[0058] In some embodiments the kits may be used in an in vitro method for characterizing a COVID-19 treatment.

[0059] In some embodiments the kits comprise a first forward primer and a first reverse primer that amplify all or part of a first SARS-CoV-2 gene selected from the group consisting of spike (S), envelope (E), membrane (M), nucleocapsid (N), accessory protein (ap) 3a (ap3a), ap3b, ap3c, ap3d, ap6, ap7a, ap7b, ap8, ap9a, ap9b, ap9c, ap10 and ap14, to form a first amplified product; and a second forward primer and a second reverse primer that amplify all or part of a second SARS-CoV-2 gene selected from the group consisting of non-structural protein (nsp) 1 (nsp1 ), nsp1 , nsp2, nsp3, nsp4, nsp5, nsp6, nsp7, nsp8, nsp9, nsp10, nsp11 , nsp12, nsp13, nsp14, nsp15 and nsp16, to form a second amplified product. In some embodiments the kits further comprise a first probe that hybridizes to the first amplified product and a second probe that hybridizes to the second amplified product. In some embodiments the first SARS-CoV-2 gene is selected from the group consisting of N, E, and ap7a. In some embodiments the second SARS- CoV-2 gene is selected from the group consisting of nsp12 and nsp13.

[0060] In some embodiments of the kits:

[0061 ] (A) when the first SARS-CoV-2 gene is N the first forward primer hybridizes to the sequence TAGCAGGAGAAGTTCCCC (SEQ ID NO: 14) and the RNA equivalent thereof; the first reverse primer hybridizes to the sequence TTGAGAGCAAAATGTCTG (SEQ ID NO: 15) and the RNA equivalent thereof; and the first probe hybridizes to the sequence AATCTGTCAAGCAGCAGCAA (SEQ ID NO: 16) and the RNA equivalent thereof;

[0062] (B) when the first SARS-CoV-2 gene is E the first forward primer hybridizes to the sequence ACGCTATTAACTATTAACGTACCTGA (SEQ ID NO: 20) and the RNA equivalent thereof; the first reverse primer hybridizes to the sequence TGTGTGCGTACTGCTGCAATAT (SEQ ID NO: 21 ) and the RNA equivalent thereof; and the first probe hybridizes to the sequence CGAAGCGCAGTAAGGATGGCTAGTGT (SEQ ID NO: 22) and the RNA equivalent thereof;

[0063] (C) when the second SARS-CoV-2 gene is nsp12 the second forward primer hybridizes to the sequence CGAAATCATACCAGTTACC (SEQ ID NO: 17) and the RNA equivalent thereof; the second reverse primer hybridizes to the sequence CCTATATTAACCTTGACCAG (SEQ ID NO: 18) and the RNA equivalent thereof; and the second probe hybridizes to the sequence CCTGGCGTGGTTTGTATGA (SEQ ID NO: 19) and the RNA equivalent thereof; and

[0064] (D) when the second SARS-CoV-2 gene is nsp13 the second forward primer hybridizes to the sequence CTGGTTCTAGTGTGCCCTTA (SEQ ID NO: 23) and the RNA equivalent thereof; the second reverse primer hybridizes to the sequence CGTTGTCCTGCTGAAATTGT (SEQ ID NO: 24) and the RNA equivalent thereof; and the second probe hybridizes to the sequence TTCCGAGGAACATGTCTGGACCT (SEQ ID NO: 25) and the RNA equivalent thereof.

[0065] In some embodiments of the kits:

[0066] (A) when the first SARS-CoV-2 gene is N the first forward primer comprises the sequence GGGGAACTTCTCCTGCTA (SEQ ID NO: 2) and the RNA equivalent thereof; the first reverse primer comprises the sequence CAGACATTTTGCTCTCAA (SEQ ID NO: 3) and the RNA equivalent thereof; and the first probe comprises the sequence TTGCTGCTGCTTGACAGATT (SEQ ID NO: 4) and the RNA equivalent thereof;

[0067] (B) when the first SARS-CoV-2 gene is E the first forward primer comprises the sequence ACAGGTACGTTAATAGTTAATAGCGT (SEQ ID NO: 8) and the RNA equivalent thereof; the first reverse primer comprises the sequence ATATTGCAGCAGTACGCACACA (SEQ ID NO: 9) and the RNA equivalent thereof; and the first probe comprises the sequence ACACTAGCCATCCTTACTGCGCTTCG (SEQ ID NO: 10) and the RNA equivalent thereof;

[0068] (C) when the second SARS-CoV-2 gene is nsp12 the second forward primer comprises the sequence GGTAACTGGTATGATTTCG (SEQ ID NO: 5) and the RNA equivalent thereof; the second reverse primer comprises the sequence CTGGTCAAGGTTAATATAGG (SEQ ID NO: 6) and the RNA equivalent thereof; and the second probe comprises the sequence TCATACAAACCACGCCAGG (SEQ ID NO: 7) and the RNA equivalent thereof; and

[0069] (D) when the second SARS-CoV-2 gene is nsp13 the second forward primer comprises the sequence TAAGGGCACACTAGAACCAG (SEQ ID NO: 1 1 ) and the RNA equivalent thereof; the second reverse primer comprises the sequence ACAATTTCAGCAGGACAACG (SEQ ID NO: 12) and the RNA equivalent thereof; and the second probe comprises the sequence AGGTCCAGACATGTTCCTCGGAA (SEQ ID NO: 13) and the RNA equivalent thereof.

[0070] In some embodiments of the kits:

[0071] (A) when the first SARS-CoV-2 gene is N the first forward primer consists of the sequence GGGGAACTTCTCCTGCTA (SEQ ID NO: 2) and the RNA equivalent thereof; the first reverse primer consists of the sequence CAGACATTTTGCTCTCAA (SEQ ID NO: 3) and the RNA equivalent thereof; and the first probe consists of the sequence TTGCTGCTGCTTGACAGATT (SEQ ID NO: 4) and the RNA equivalent thereof;

[0072] (B) when the first SARS-CoV-2 gene is E the first forward primer consists of the sequence ACAGGTACGTTAATAGTTAATAGCGT (SEQ ID NO: 8) and the RNA equivalent thereof; the first reverse primer consists of the sequence ATATTGCAGCAGTACGCACACA (SEQ ID NO: 9) and the RNA equivalent thereof; and the first probe consists of the sequence ACACTAGCCATCCTTACTGCGCTTCG (SEQ ID NO: 10) and the RNA equivalent thereof; [0073] (C) when the second SARS-CoV-2 gene is nsp12 the second forward primer consists of the sequence GGTAACTGGTATGATTTCG (SEQ ID NO: 5) and the RNA equivalent thereof; the second reverse primer consists of the sequence CTGGTCAAGGTTAATATAGG (SEQ ID NO: 6) and the RNA equivalent thereof; and the second probe consists of the sequence TCATACAAACCACGCCAGG (SEQ ID NO: 7) and the RNA equivalent thereof; and

[0074] (D) when the second SARS-CoV-2 gene is nsp13 the second forward primer consists of the sequence TAAGGGCACACTAGAACCAG (SEQ ID NO: 11 ) and the RNA equivalent thereof; the second reverse primer consists of the sequence ACAATTTCAGCAGGACAACG (SEQ ID NO: 12) and the RNA equivalent thereof; and the second probe consists of the sequence AGGTCCAGACATGTTCCTCGGAA (SEQ ID NO: 13) and the RNA equivalent thereof.

[0075] In some embodiments the kits further comprise an internal negative control, and/or an internal positive control, and/or a reverse transcriptase, and/or a DNA polymerase.

[0076] In some embodiments the kits may be used in one or more of the methods disclosed herein.

[0077] Uses

[0078] In another aspect this invention provides a set of oligonucleotides or a kit for use in characterizing in vitro the replicative activity of SARS-CoV-2 in a sample. The use comprises providing at least one subject sample; subjecting the at least one sample to a reverse transcription reaction to generate a cDNA copy of RNA in the at least one sample; amplifying any resultant cDNA formed using a first forward primer and a first reverse primer that amplify all or part of a first SARS-CoV-2 gene selected from the group consisting of spike (S), envelope (E), membrane (M), nucleocapsid (N), accessory protein (ap) 3a (ap3a), ap3b, ap3c, ap3d, ap6, ap7a, ap7b, ap8, ap9a, ap9b, ap9c, ap10 and ap14, to form a first amplified product; detecting any first amplified product formed using a first probe that hybridizes to the first amplified product; amplifying any resultant cDNA formed using a second forward primer and a second reverse primer that amplify all or part of a second SARS-CoV-2 gene selected from the group consisting of non- structural protein (nsp) 1 (nsp1 ), nsp1 , nsp2, nsp3, nsp4, nsp5, nsp6, nsp7, nsp8, nsp9, nsp10, nsp11 , nsp12, nsp13, nsp14, nsp15 and nsp16, to form a second amplified product; detecting any second amplified product formed using a second probe that hybridizes to the second amplified product; quantifying the RNA transcripts of the first SARS-CoV-2 gene in the at least one sample and quantifying the RNA transcripts of the second SARS-CoV-2 gene in the at least one sample; and calculating the ratio of the quantified RNA transcripts of the first SARS-CoV-2 gene to the quantified RNA transcripts of the second SARS-CoV-2 gene and comparing the calculated ratio to a threshold value; wherein, when the value of the ratio is higher than a threshold value, the SARS-CoV-2 is replicating in the sample. In some embodiments the first SARS-CoV- 2 gene is selected from the group consisting of N, E, and ap7a. In some embodiments the second SARS-CoV-2 gene is selected from the group consisting of nsp12 and nsp13.

[0079] In some embodiments of the oligonucleotides or a kit for use:

[0080] (A) when the first SARS-CoV-2 gene is N the first forward primer hybridizes to the sequence TAGCAGGAGAAGTTCCCC (SEQ ID NO: 14) and the RNA equivalent thereof; the first reverse primer hybridizes to the sequence TTGAGAGCAAAATGTCTG (SEQ ID NO: 15) and the RNA equivalent thereof; and the first probe hybridizes to the sequence AATCTGTCAAGCAGCAGCAA (SEQ ID NO: 16) and the RNA equivalent thereof;

[0081 ] (B) when the first SARS-CoV-2 gene is E the first forward primer hybridizes to the sequence ACGCTATTAACTATTAACGTACCTGA (SEQ ID NO: 20) and the RNA equivalent thereof; the first reverse primer hybridizes to the sequence TGTGTGCGTACTGCTGCAATAT (SEQ ID NO: 21 ) and the RNA equivalent thereof; and the first probe hybridizes to the sequence CGAAGCGCAGTAAGGATGGCTAGTGT (SEQ ID NO: 22) and the RNA equivalent thereof; [0082] (C) when the second SARS-CoV-2 gene is nsp12 the second forward primer hybridizes to the sequence CGAAATCATACCAGTTACC (SEQ ID NO: 17) and the RNA equivalent thereof; the second reverse primer hybridizes to the sequence CCTATATTAACCTTGACCAG (SEQ ID NO: 18) and the RNA equivalent thereof; and the second probe hybridizes to the sequence CCTGGCGTGGTTTGTATGA (SEQ ID NO: 19) and the RNA equivalent thereof; and

[0083] (D) when the second SARS-CoV-2 gene is nsp13 the second forward primer hybridizes to the sequence CTGGTTCTAGTGTGCCCTTA (SEQ ID NO: 23) and the RNA equivalent thereof; the second reverse primer hybridizes to the sequence CGTTGTCCTGCTGAAATTGT (SEQ ID NO: 24) and the RNA equivalent thereof; and the second probe hybridizes to the sequence TTCCGAGGAACATGTCTGGACCT (SEQ ID NO: 25) and the RNA equivalent thereof.

[0084] In some embodiments of the oligonucleotides or a kit for use:

[0085] (A) when the first SARS-CoV-2 gene is N the first forward primer comprises the sequence GGGGAACTTCTCCTGCTA (SEQ ID NO: 2) and the RNA equivalent thereof; the first reverse primer comprises the sequence CAGACATTTTGCTCTCAA (SEQ ID NO: 3) and the RNA equivalent thereof; and the first probe comprises the sequence TTGCTGCTGCTTGACAGATT (SEQ ID NO: 4) and the RNA equivalent thereof;

[0086] (B) when the first SARS-CoV-2 gene is E the first forward primer comprises the sequence ACAGGTACGTTAATAGTTAATAGCGT (SEQ ID NO: 8) and the RNA equivalent thereof; the first reverse primer comprises the sequence ATATTGCAGCAGTACGCACACA (SEQ ID NO: 9) and the RNA equivalent thereof; and the first probe comprises the sequence ACACTAGCCATCCTTACTGCGCTTCG (SEQ ID NO: 10) and the RNA equivalent thereof;

[0087] (C) when the second SARS-CoV-2 gene is nsp12 the second forward primer comprises the sequence GGTAACTGGTATGATTTCG (SEQ ID NO: 5) and the RNA equivalent thereof; the second reverse primer comprises the sequence CTGGTCAAGGTTAATATAGG (SEQ ID NO: 6) and the RNA equivalent thereof; and the second probe comprises the sequence TCATACAAACCACGCCAGG (SEQ ID NO: 7) and the RNA equivalent thereof; and

[0088] (D) when the second SARS-CoV-2 gene is nsp13 the second forward primer comprises the sequence TAAGGGCACACTAGAACCAG (SEQ ID NO: 1 1 ) and the RNA equivalent thereof; the second reverse primer comprises the sequence ACAATTTCAGCAGGACAACG (SEQ ID NO: 12) and the RNA equivalent thereof; and the second probe comprises the sequence AGGTCCAGACATGTTCCTCGGAA (SEQ ID NO: 13) and the RNA equivalent thereof.

[0089] In some embodiments of the oligonucleotides or a kit for use:

[0090] (A) when the first SARS-CoV-2 gene is N the first forward primer consists of the sequence GGGGAACTTCTCCTGCTA (SEQ ID NO: 2) and the RNA equivalent thereof; the first reverse primer consists of the sequence CAGACATTTTGCTCTCAA (SEQ ID NO: 3) and the RNA equivalent thereof; and the first probe consists of the sequence TTGCTGCTGCTTGACAGATT (SEQ ID NO: 4) and the RNA equivalent thereof;

[0091] (B) when the first SARS-CoV-2 gene is E the first forward primer consists of the sequence ACAGGTACGTTAATAGTTAATAGCGT (SEQ ID NO: 8) and the RNA equivalent thereof; the first reverse primer consists of the sequence ATATTGCAGCAGTACGCACACA (SEQ ID NO: 9) and the RNA equivalent thereof; and the first probe consists of the sequence ACACTAGCCATCCTTACTGCGCTTCG (SEQ ID NO: 10) and the RNA equivalent thereof;

[0092] (C) when the second SARS-CoV-2 gene is nsp12 the second forward primer consists of the sequence GGTAACTGGTATGATTTCG (SEQ ID NO: 5) and the RNA equivalent thereof; the second reverse primer consists of the sequence CTGGTCAAGGTTAATATAGG (SEQ ID NO: 6) and the RNA equivalent thereof; and the second probe consists of the sequence TCATACAAACCACGCCAGG (SEQ ID NO: 7) and the RNA equivalent thereof; and

[0093] (D) when the second SARS-CoV-2 gene is nsp13 the second forward primer consists of the sequence TAAGGGCACACTAGAACCAG (SEQ ID NO: 11 ) and the RNA equivalent thereof; the second reverse primer consists of the sequence ACAATTTCAGCAGGACAACG (SEQ ID NO: 12) and the RNA equivalent thereof; and the second probe consists of the sequence AGGTCCAGACATGTTCCTCGGAA (SEQ ID NO: 13) and the RNA equivalent thereof.

[0094] In some embodiments of the oligonucleotides or a kit for use, amplification of the first amplified product using the first forward primer and the first reverse primer is performed in a first container and amplification of the second amplified product using the second forward primer and the second reverse primer is performed in a second container.

[0095] In some embodiments of the oligonucleotides or a kit for use, amplification of the first amplified product using the first forward primer and the first reverse primer, and amplification of the second amplified product using the second forward primer and the second reverse primer are performed in the same container.

[0096] In another aspect this invention provides the use of a set of oligonucleotides or a kit for characterizing in vitro the replicative activity of SARS-CoV-2 in a sample. The use comprises providing at least one subject sample; subjecting the at least one sample to a reverse transcription reaction to generate a cDNA copy of RNA in the at least one sample; amplifying any resultant cDNA formed using a first forward primer and a first reverse primer that amplify all or part of a first SARS-CoV-2 gene selected from the group consisting of spike (S), envelope (E), membrane (M), nucleocapsid (N), accessory protein (ap) 3a (ap3a), ap3b, ap3c, ap3d, ap6, ap7a, ap7b, ap8, ap9a, ap9b, ap9c, ap10 and ap14, to form a first amplified product; detecting any first amplified product formed using a first probe that hybridizes to the first amplified product; amplifying any resultant cDNA formed using a second forward primer and a second reverse primer that amplify all or part of a second SARS-CoV-2 gene selected from the group consisting of non-structural protein (nsp) 1 (nsp1 ), nsp1 , nsp2, nsp3, nsp4, nsp5, nsp6, nsp7, nsp8, nsp9, nsp10, nsp11 , nsp12, nsp13, nsp14, nsp15 and nsp16, to form a second amplified product; detecting any second amplified product formed using a second probe that hybridizes to the second amplified product; quantifying the RNA transcripts of the first SARS-CoV-2 gene in the at least one sample and quantifying the RNA transcripts of the second SARS-CoV-2 gene in the at least one sample; and calculating the ratio of the quantified RNA transcripts of the first SARS-CoV- 2 gene to the quantified RNA transcripts of the second SARS-CoV-2 gene and comparing the calculated ratio to a threshold value; wherein, when the value of the ratio is higher than a threshold value, the SARS-CoV-2 is replicating in the sample. In some embodiments the first SARS-CoV-2 gene is selected from the group consisting of N, E, and ap7a. In some embodiments the second SARS-CoV-2 gene is selected from the group consisting of nsp12 and nsp13.

[0097] In some embodiments of the use:

[0098] (A) when the first SARS-CoV-2 gene is N the first forward primer hybridizes to the sequence TAGCAGGAGAAGTTCCCC (SEQ ID NO: 14) and the RNA equivalent thereof; the first reverse primer hybridizes to the sequence TTGAGAGCAAAATGTCTG (SEQ ID NO: 15) and the RNA equivalent thereof; and the first probe hybridizes to the sequence AATCTGTCAAGCAGCAGCAA (SEQ ID NO: 16) and the RNA equivalent thereof;

[0099] (B) when the first SARS-CoV-2 gene is E the first forward primer hybridizes to the sequence ACGCTATTAACTATTAACGTACCTGA (SEQ ID NO: 20) and the RNA equivalent thereof; the first reverse primer hybridizes to the sequence TGTGTGCGTACTGCTGCAATAT (SEQ ID NO: 21 ) and the RNA equivalent thereof; and the first probe hybridizes to the sequence CGAAGCGCAGTAAGGATGGCTAGTGT (SEQ ID NO: 22) and the RNA equivalent thereof; [0100] (C) when the second SARS-CoV-2 gene is nsp12 the second forward primer hybridizes to the sequence CGAAATCATACCAGTTACC (SEQ ID NO: 17) and the RNA equivalent thereof; the second reverse primer hybridizes to the sequence CCTATATTAACCTTGACCAG (SEQ ID NO: 18) and the RNA equivalent thereof; and the second probe hybridizes to the sequence CCTGGCGTGGTTTGTATGA (SEQ ID NO: 19) and the RNA equivalent thereof; and

[0101] (D) when the second SARS-CoV-2 gene is nsp13 the second forward primer hybridizes to the sequence CTGGTTCTAGTGTGCCCTTA (SEQ ID NO: 23) and the RNA equivalent thereof; the second reverse primer hybridizes to the sequence CGTTGTCCTGCTGAAATTGT (SEQ ID NO: 24) and the RNA equivalent thereof; and the second probe hybridizes to the sequence TTCCGAGGAACATGTCTGGACCT (SEQ ID NO: 25) and the RNA equivalent thereof.

[0102] In some embodiments of the use:

[0103] (A) when the first SARS-CoV-2 gene is N the first forward primer comprises the sequence GGGGAACTTCTCCTGCTA (SEQ ID NO: 2) and the RNA equivalent thereof; the first reverse primer comprises the sequence CAGACATTTTGCTCTCAA (SEQ ID NO: 3) and the RNA equivalent thereof; and the first probe comprises the sequence TTGCTGCTGCTTGACAGATT (SEQ ID NO: 4) and the RNA equivalent thereof;

[0104] (B) when the first SARS-CoV-2 gene is E the first forward primer comprises the sequence ACAGGTACGTTAATAGTTAATAGCGT (SEQ ID NO: 8) and the RNA equivalent thereof; the first reverse primer comprises the sequence ATATTGCAGCAGTACGCACACA (SEQ ID NO: 9) and the RNA equivalent thereof; and the first probe comprises the sequence ACACTAGCCATCCTTACTGCGCTTCG (SEQ ID NO: 10) and the RNA equivalent thereof;

[0105] (C) when the second SARS-CoV-2 gene is nsp12 the second forward primer comprises the sequence GGTAACTGGTATGATTTCG (SEQ ID NO: 5) and the RNA equivalent thereof; the second reverse primer comprises the sequence CTGGTCAAGGTTAATATAGG (SEQ ID NO: 6) and the RNA equivalent thereof; and the second probe comprises the sequence TCATACAAACCACGCCAGG (SEQ ID NO: 7) and the RNA equivalent thereof; and

[0106] (D) when the second SARS-CoV-2 gene is nsp13 the second forward primer comprises the sequence TAAGGGCACACTAGAACCAG (SEQ ID NO: 1 1 ) and the RNA equivalent thereof; the second reverse primer comprises the sequence ACAATTTCAGCAGGACAACG (SEQ ID NO: 12) and the RNA equivalent thereof; and the second probe comprises the sequence AGGTCCAGACATGTTCCTCGGAA (SEQ ID NO: 13) and the RNA equivalent thereof.

[0107] In some embodiments of the use:

[0108] (A) when the first SARS-CoV-2 gene is N the first forward primer consists of the sequence GGGGAACTTCTCCTGCTA (SEQ ID NO: 2) and the RNA equivalent thereof; the first reverse primer consists of the sequence CAGACATTTTGCTCTCAA (SEQ ID NO: 3) and the RNA equivalent thereof; and the first probe consists of the sequence TTGCTGCTGCTTGACAGATT (SEQ ID NO: 4) and the RNA equivalent thereof;

[0109] (B) when the first SARS-CoV-2 gene is E the first forward primer consists of the sequence ACAGGTACGTTAATAGTTAATAGCGT (SEQ ID NO: 8) and the RNA equivalent thereof; the first reverse primer consists of the sequence ATATTGCAGCAGTACGCACACA (SEQ ID NO: 9) and the RNA equivalent thereof; and the first probe consists of the sequence ACACTAGCCATCCTTACTGCGCTTCG (SEQ ID NO: 10) and the RNA equivalent thereof;

[0110] (C) when the second SARS-CoV-2 gene is nsp12 the second forward primer consists of the sequence GGTAACTGGTATGATTTCG (SEQ ID NO: 5) and the RNA equivalent thereof; the second reverse primer consists of the sequence CTGGTCAAGGTTAATATAGG (SEQ ID NO: 6) and the RNA equivalent thereof; and the second probe consists of the sequence TCATACAAACCACGCCAGG (SEQ ID NO: 7) and the RNA equivalent thereof; and

[0111] (D) when the second SARS-CoV-2 gene is nsp13 the second forward primer consists of the sequence TAAGGGCACACTAGAACCAG (SEQ ID NO: 11 ) and the RNA equivalent thereof; the second reverse primer consists of the sequence ACAATTTCAGCAGGACAACG (SEQ ID NO: 12) and the RNA equivalent thereof; and the second probe consists of the sequence AGGTCCAGACATGTTCCTCGGAA (SEQ ID NO: 13) and the RNA equivalent thereof.

[0112] In some embodiments of the use, amplification of the first amplified product using the first forward primer and the first reverse primer is performed in a first container and amplification of the second amplified product using the second forward primer and the second reverse primer is performed in a second container.

[0113] In some embodiments of the use, amplification of the first amplified product using the first forward primer and the first reverse primer, and amplification of the second amplified product using the second forward primer and the second reverse primer are performed in the same container.

BRIEF DESCRIPTION OF THE DRAWINGS

[0114] Figure 1 : Representative examples of single-step (RT+ddPCR) amplification of SARS-CoV-2 RNA.

[0115] Figure 2: (A) Flow cytometric analysis of A549-ACE2 and (B) Calu-3 cells infected with SARS-CoV-2. A549-ACE2 or Calu-3 cells were infected at MOI 0.3 for 24 hours. Surface S staining of infected cells was analyzed by flow cytometry. Results are representative of two independent experiments. The percentage of cells positive for the viral spike protein is indicated in the top right corner. (C) Normalized detection rates by ddPCR of different targets on SARS-CoV-2. The number of positive droplets obtained by RdRP-IP4 amplification was taken as reference. Viral particles stocks were obtained from Vero cells. [0116] Figure 3: (A) The outcome of ddPCR on three categories of samples as initially scored for SARS-CoV-2 RNA presence by qRT-PCR. (B) Comparison of quantification cycles (Cq) for RACK1 mRNA in airways RNA samples from ddPCR positive and ddPCR negative samples.

[0117] Figure 4: (A) Age of patients according to ddPCR outcome. (B) Prevalence of few clinical symptoms according to ddPCR outcome.

[0118] Figure 5: Comparison of RNA extraction methods. Concentrations (A) and quantities (B) of RNA obtained from naso-pharyngeal swabs after Tri-reagent (phenol+guanidinium thiocyanate, Y axis) or Nucleospin extraction (column, X axis). C- D; ddPCR amplification of E (Y axis) and IP4 (X axis) from SARS-CoV-2 on the same volume RNA sample. E; droplets quantification from experiments C and D.

[0119] Figure 6: Representative examples of reverse transcription (RT) optimization applied to 2-steps ddPCR on E and IP4 amplimeres on SARS-CoV-2 RNA. The names of the kits tested are mentioned above each figure. All kits used random priming. Addition of specific SARS-CoV-2 primers is mentioned above the figure. 5 pL of the same RNA was used for each RT in a final volume of 20 pL. ddPCR was performed on 5 pL of RT. Primers and probe concentration are mentioned in Table 3. Outcome of the comparison is shown on bar-chart and express in copy of target per droplet (cpd).

[0120] Figure 7: Quantification of four candidate housekeeping genes on nine naso-pharyngeal swab samples. RACK1 (receptor for activated C kinase 1 ) mRNA was the most expressed (lower Cq) and chosen to assess clinical sample quality.

[0121] Figure 8: Comparison of Bio-rad SARS-CoV-2 ddPCR kit (dEXD28563542, Cat#1200802, left panels) and in-house developed ddPCR assays on three clinical samples (right panels). Bio-Rad assay targets two different segments of N gene and RPP30 mRNA as a control while our test associates N and IP4-RdRP. The dividing lines separating droplets with different content are sometimes difficult to draw with the commercial kits. DETAILED DESCRITION

[0122] RT-qPCR detection of SARS-CoV-2 RNA still represents the method of reference to diagnose and monitor COVID-19. From the pandemic onset, however, doubts have been expressed concerning the sensitivity of this molecular diagnosis. Droplet digital PCR is a third-generation PCR technique particularly adapted to detect low biotic burdens. We developed two-colors ddPCR assays for the detection of 4 different regions of SARS-CoV-2 RNA including non-structural protein-encoding (IP4- RdRP, helicase) and structural ones (E, N). We observed that N or E gene sequences are commonly more abundant than IP4 and helicase in cells infected in vitro suggesting that detection of N gene, encoded by the most abundant sub-genomic RNA of SARS- CoV-2, will increase the sensitivity of detection during the highly replicative phase of infection. We investigated 208 nasopharyngeal swabs sampled in March-April 2020 in different hospitals of the Greater Paris. We observed that 5.9% of informative samples (n=11/185) initially scored as “non-positive” by RT-qPCR were positive for at least two targets on SARS-CoV-2 RNA with ddPCR. Our work confirms that the use of ddPCR increases the proportion of positive samples in upper airways, albeit in a rather limited proportion in the frame of the first line diagnosis on a French population. Systematic targeting of the N gene of SARS-CoV-2 appears as a reasonable and simple improvement in COVID-19 diagnosis.

A. SARS-CoV-2 Nucleic Acids

[0123] As used herein a “variant” of a reference sequence of nucleotides is a modified form in which at least one nucleotide is added, deleted, or substituted. In some embodiments the variant includes only addition of one or more nucleotides. In some embodiments the variant includes only deletion of one or more nucleotides. In some embodiments the variant includes only substitution of one or more nucleotides. In some embodiments the variant includes addition and deletion of different nucleotides. An addition is a change that increases the total number of nucleotides in the sequence while a deletion is a change that decreases the total number of nucleotides. In some embodiments the addition and/or deletion occurs at only one end while in other embodiments it occurs at both ends. In some embodiments an addition or deletion is internal. In some embodiments the variant includes only one nucleotide that is added, deleted, or substituted. In some embodiments 2, 3, 4, 5, 6, 7, 8, 9 or 10 or more nucleotides are added, deleted, or substituted. A variant according to the invention hybridizes to SARS-CoV-2 nucleic acid (RNA, DNA equivalent or complement thereof). In this context, the term “hybridizes to” refers to the ability of the variant to form a doublestranded hybrid molecule with SARS-CoV-2 nucleic acid.

[0124] According to standard practice in the field of virology, the sequences of coronavirus genome (positive single stranded RNA) or fragments thereof (target sequences for SARS-CoV-2 RNA amplification) are disclosed in the DNA form. Therefore, the sequence SEQ ID NO: 1 is the DNA equivalent of SARS-CoV-2 RNA.

[0125] SEQ ID NO: 1 is the severe acute respiratory syndrome coronavirus 2 isolate Wuhan-Hu-1 , complete genome. This sequence is in the NCBI database as NCBI Reference Sequence: NC_045512.2. SEQ ID NO: 1 is 29,903 nucleotides in length.

[0126] As used herein a “SARS-CoV-2 gene” refers to an open reading frame that encodes a SARS-CoV-2 protein.

[0127] Based on SEQ ID NO: 1 , primers and probes were designed or selected to target the N gene, the E gene, the nsp12 gene, and the nsp13 gene. The locations of the primers and probes in the SARC-CoV-2 genome are show in Table 1 . All nucleotide positions are relative to SEQ ID NO: 1. For simplicity, the positions are defined relative to the positive strand, even though the reverse primer is a negative strand sequence. TABLE 1

[0128] The following Table 2 lists preferred embodiments of SARS-CoV-2 nucleic acid sequences of this disclosure, which are described in the examples. The primer/probe column lists the sequences of the forward primer, reverse primer, and probe used in the Examples. The Target Sequence (reverse complement) column lists the reverse complement (i.e., target) of the primer/probe sequence. The sequences are represented as DNA but in alternative embodiments at least one of the nucleotides may be an RNA nucleotide.

Table 2

Methods For Detection

[0129] The invention encompasses methods for specific detection of SARS-CoV- 2. In one embodiment, the method comprises providing a sample, subjecting the sample to a reverse transcription reaction to generate a cDNA copy SARS-CoV-2 RNA in the sample using a “reverse primer” specific for coronavirus, amplifying any resultant DNA with the “reverse primer” and a “forward primer,” and detecting any amplified product with a “probe.” The method can be used for the determination of whether or not SARS- CoV-2 is present in the sample.

[0130] The sample is a biological sample, for example, tissue, body fluid, or stool. In a preferred embodiment the sample is selected from saliva, sputum, induced sputa (IS), and upper respiratory specimens (URS). In another preferred embodiment the sample is selected from oral, nasal, oropharyngeal and nasopharyngeal (NP) swabs, aspirate, wash and/or lavage. In another preferred embodiment the sample is selected from tracheal aspirate and bronchoalveolar lavage (BAL).

[0131] The sample can be subjected to well-known isolation and purification protocols or used directly. For example, the sample can be subjected to a treatment to release/extract the nucleic acids of the sample and/or to remove proteins and other non- nucleic acid components of the sample using conventional techniques, such as those in the Examples.

[0132] Reverse transcription of the RNA of a coronavirus strain can be performed with a “reverse primer” specific for coronavirus. A “reverse primer” is one that, based on its 5’-3’ orientation, can bind to a single-stranded RNA and serve to initiate generation of a complementary DNA (cDNA) copy of the RNA. The reverse transcription can be accomplished using well known and routine methods. The reaction mix for reverse transcription contains the reagents for the reaction, for example, a reverse primer, dNTPs (dATP, dCTP, dGTP and dTTP), a buffer, and a reverse transcriptase. [0133] Reverse transcription of the RNA of a coronavirus strain can alternatively be performed using an oligo-dT primer and/or random primers.

[0134] Exemplary reaction conditions for reverse transcription are set forth in the examples.

[0135] Amplification of the cDNA copy of a coronavirus strain generated by reverse transcription can be performed with a “forward primer” specific for coronavirus. A “forward primer” is one that, based on its 5’-3’ orientation, can bind to a single-stranded antisense cDNA copy of an RNA generated by reverse transcription and serve to initiate generation of a double-stranded DNA copy of the RNA. The amplification can be accomplished using well known and routine methods. The reagent mix for amplification contains the reagents for the reaction, for example a forward primer, a reverse primer, dNTPs, a buffer, and a DNA polymerase.

[0136] In one embodiment, the method of the invention is performed using a single RT-PCR reagent mix containing the reagents for the reverse transcription and amplification reactions. Preferably, the reverse primer used for the reverse transcription reaction is also used for the amplification reaction.

[0137] Preferably, the reverse transcription and amplification reactions are performed in a plastic or glass container, most preferably in the same container.

[0138] Amplification methods known in the art include RCA, MDA, NASBA, TMA, SDA, LCR, b-DNA, PCR (all forms including RT-PCR), RAM, LAMP, ICAN, SPIA, QB- replicase, or Invader. A preferred amplification method is the polymerase chain reaction (PCR) amplification. See, e.g., PCR Technology: Principles and Applications for DNA Amplification (Ed. H. A. Erlich, Freeman Press, NY, N.Y., 1992); PCR Protocols: A Guide to Methods and Applications (Eds. linis, et al., Academic Press, San Diego, Calif., 1990); Mattila et al., Nucleic Acids Res. 19, 4967 (1991 ); Eckert et al., PCR Methods and Applications 1 , 17 (1991 ); PCR (Eds. McPherson et al., IRL Press, Oxford); and U.S. Pat. Nos. 4,683,202, 4,683,195, 4,800,159 4,965,188, and 5,333,675. More preferred PCR methods is real-time PCR, PCR-HRM (High-Resolution DNA Melting) (see Andriantsoanirina et al. Journal of Microbiological Methods, 78 : 165 (2009)) and PCR coupled to ligase detection reaction based on fluorescent microsphere (Luminex® microspheres).

[0139] Amplification techniques include in particular isothermal methods and PCR-based techniques. Isothermal techniques include such methods as nucleic acid sequence-based amplification (NASBA), loop-mediated isothermal amplification (LAMP), helicase-dependent amplification (HDA), rolling circle amplification (RCA), and strand displacement amplification (SDA), exponential amplification reaction (EXPAR), isothermal and chimeric primer-initiated amplification of nucleic acids (ICANs), signal- mediated amplification of RNA technology (SMART) and others (see e.g. Asiello and Baeumner, Lab Chip; 11 (8): 1420-1430, 2011 ).

[0140] Preferably, the PCR technique quantitatively measures starting amounts of DNA, cDNA, or RNA. Examples of PCR-based techniques according to the invention include techniques such as, but not limited to, quantitative PCR (Q-PCR), reversetranscriptase polymerase chain reaction (RT-PCR), quantitative reverse-transcriptase PCR (QRT-PCR), or digital PCR. These techniques are well known and easily available technologies for those skilled in the art.

[0141] Preferably, the method is a one-step real-time RT-PCR assay, for example, as described in the Examples.

[0142] Preferably, a probe is used to detect the amplified product. The probe can be labeled with a fluorescent, radioactive, or enzymatic label. The amplified product can be detected with a specific detection chemistry such as fluorescence resonance energy transfer (FRET) probes, TAQMAN probes, molecular beacons, scorpion probes, fluorescently labeled (or other labeled) primers, lightup probes or a dye-based chemistry, DNA, PNA, LNA, or RNA including modified bases that bind to the amplified product to detect the sequence of interest. [0143] Detection of the amplified products can be real-time (during the amplification process) or endpoint (after the amplification process). The invention allows for detection of the amplification products in the same vessel as amplification occurs.

[0144] Preferably, a DNA internal control is used to monitor the amplification reaction.

[0145] Preferably, a RNA internal control is used to monitor the reverse transcription and amplification reactions.

[0146] In some embodiments the amplification is performed using a quantitative PGR (q-PCR) technique.

[0147] In some embodiments the amplification is performed using a real-time PGR

(RT-PCR) technique.

[0148] In a preferred embodiment the amplification is performed using a droplet digital PGR (ddPCR) technique. Exemplary reagents and techniques are disclosed in the examples.

[0149] The “forward primer” is a sense primer. In some embodiments the forward primer is specific for a subset of SARS-CoV-2. In some embodiments the forward primer is specific for SARS-CoV-2.

[0150] The “reverse primer” is an anti-sense primer. In some embodiments the reverse primer is specific for a subset of SARS-CoV-2. In some embodiments the reverse primer is specific for SARS-CoV-2.

[0151] In some embodiments the forward primer and/or reverse primer are defined by the feature that they hybridize to a defined sequence target. In this context, the term “hybridizes to” refers to the ability of the primer to form a double-stranded hybrid molecule with SARS-CoV-2 RNA (i.e., comprising the RNA equivalent of all or part of SEQ ID NO: 1 ) sufficient to produce a cDNA and/or to promote amplification of a cDNA under standard reverse transcription and amplification conditions such as those set forth in the examples.

[0152] In various embodiments, the forward primer and the reverse primer are independently at least 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in length. In various embodiments, the forward primer and the reverse primer are independently 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in length.

Probes

[0153] The probes of the invention are useful for detection of coronavirus nucleic acids. As referred to herein, the “probe” of the invention is linked to a detectable label suitable for use in a method the invention. In some embodiments the probe is specific for a subset of SARS-CoV-2 strains. In some embodiments the probe is specific for SARS-CoV-2.

[0154] A “detectable label” as used herein is a moiety, which can be detected directly or indirectly. In some embodiments, detection of the label involves directly detecting an emission of energy by the label (e.g., radioactivity, luminescence, optical). A label can also be detected indirectly by its ability to bind to or cleave another moiety, which itself may emit or absorb light of a particular wavelength (e.g., biotin, avidin, epitope tag such as the FLAG epitope, enzyme tag such as horseradish peroxidase). Preferred detectable labels include radioactive labels, fluorescent labels, chemiluminescent labels, bioluminescent labels, and epitope tags. Preferably, the probe is labelled with the fluorescent dyes 6-carboxy-fluorescein (6FAM) or hexachloro-6- carboxy-fluorescein (HEX), most preferably at the 5 ’end. Preferably, the probe is labelled at its 3’ end with black hole quencher 1 (BHQ1 ).

[0155] In various embodiments, the probe is at least 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in length. In various embodiments, the probe is 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in length.

Controls

[0156] In various embodiments, the invention encompasses the inclusion of controls for the reverse transcription and/or amplification reactions. The DNA control of the invention is useful to monitor the amplification reaction.

[0157] In various embodiments, the control is an internal positive control.

[0158] In some embodiments, a real-time RT-PCR assay includes in addition of unknown samples:

[0159] Two negative samples bracketing unknown samples during RNA extraction (negative extraction controls); and/or

[0160] Positive controls (in duplicate); when using in vitro synthesized transcripts as controls include five quantification positive controls (in duplicate) including 10⁵, 10⁴ and 10³ copies genome equivalent (ge) of in vitro synthesized RNA transcripts; and/or one negative amplification control.

Kits

[0161] The kits of the invention are useful for the amplification of segments of nucleic acid having a sequence complementary to a forward primer at one end and a sequence complementary to a reverse primer at the other end. The kits may also be useful for reverse transcribing viral RNA to produce cDNA. The kits can contain reagents for each of these reactions. The kits of the invention can contain any of the primers, controls, and probes of the invention, alone or in any and all combinations.

[0162] In various embodiments, the kit comprises buffer(s), a reverse transcriptase, a DNA polymerase, dNTPs, primer(s), probe(s), and/or an internal control (s). Examples

Example 1 : Materials and Methods

Samples

[0163] Collected samples were obtained in the frame of routine medical investigations and all patients were primarily processed through the state of the art of diagnostic procedure established for COVID-19. In addition, necropsy samples from three patients who died from COVID-19-associated encephalopathy were analyzed. Different brain territories were sampled in each case: cranial nerves (olfactory bulb and tract, trigeminal nucleus), brain stem (medulla oblongata, periaqueductal gray), diencephalon (hypothalamus), cerebrum (splenium corpus callosum, middle frontal, and middle temporal gyri). See Table 3.

RNA extraction

[0164] Following sampling, nasopharyngeal swabs of suspected cases of COVID- 19 were stored in sterile vials and soaked in a 1.5mL viral transport medium (VTM). A volume of 100 to 400 pL VTM was extracted either with Tri-Reagent LS (MRC, Cincinnati, OH, USA) or with Nucleospin DX Virus viral nucleic acid isolation kit (Macherey-Nagel, Duren, Germany) according to manufacturer instructions. In the case of Tri-Reagent LS extraction, RNA precipitation was performed in presence of 4pL Glycogen (5pg/pL, Ambion, Austin, TX, USA). Tri-Reagent-extracted RNA was resuspended in 20 pL RNAse-free water (Ambion) in presence of 1 pL SUPERase-IN (20U/pL, InVitrogen, Carlsbad, CA, USA) while Nucleospin-produced RNA was resuspended in 50 pL RNAse-free water. RNA concentrations were measured with Qubit RNA HS kit (Thermo Fisher Scientific, lllkirch-Graffenstaden, France). They were ranging from 1.4 to 10 ng/mL in initial suspensions. The two extraction methods employed yielded similar outcomes concerning the quality of RNA. RNA quantities were significantly higher with the column-based method albeit at the expense of RNA concentrations a crucial aspect of SARS-CoV-2 detection (see Figure 5A-B).

Quantitative Real-Time-PCR

[0165] QRT-PCR was performed according to a single-step procedure using GoScript RT mix and GoTaq probe PCR master mix (Promega, Charbonnieres, France) in the same well with 400nM of each primer and 200nM of probe. The two viral targets were RdRP-IP4 and E (8, 20). According to the WHO recommendations, confirmed positive cases were patients with both targets positive.

[0166] Out of the 252 samples investigated, 228 (90.4%) were considered as negative after qPCR, 8 (3.1%) as inconclusive, and 16 (6.3%) were scored positive for SARS-Cov-2 RNA by qPCR. cDNA synthesis.

[0167] Several reverse transcription kits were used according to manufacturer instructions. The comparison included SuperScript III (InVitrogen), Superscript IV (InVitrogen), high capacity RT kit (Applied Biosystems, Foster City, CA, USA), Prime Script (Takara Bio, Kusatsu, Japan), iSCript (Bio-Rad, Hercules, CA, USA), and iSCript advanced (Bio-Rad). RNA volumes ranging between 5 and 14.8 pL were used for reverse transcription. With regard to the two-step procedure that uses independent reverse transcription and subsequent ddPCR, among the six RT kits used, iScript™ Advanced (Bio-Rad) produced consistently larger numbers of positive droplets when tested on dilutions (in distilled water or in human cell line RNA solution corresponding to median concentrations obtained in swab samples ie 2.5ng/pL) of a positive sample for SARS-CoV-2. The RT step was adapted to each target through the addition of random, oligo-dT and virus-specific primers in different combinations and/or concentrations to increase ddPCR yields (See Table 4 and Figure 6)

[0168] Finally, to remain relevant to procedures generally used in clinical laboratories, we employed the one-step RT-ddPCR kit (Bio-Rad) corresponding to a single step version of iScript™ Advanced. One of its major advantage is to allow the direct addition of 10 pL of extracted RNA in the ddPCR, thus increasing the probability to detect the virus by comparison with 2-steps procedure that allow the addition of a maximum equivalent of 2.5 pL of RNA.

Droplet digital PCR

[0169] Most of the primers and probes employed have been published elsewhere (Table 4) (8, 20, 21 ). Four viral targets (envelop, E, nucleocapsid, N, RNA-dependent RNA polymerase, RdRP-nsp12, and Helicase-nsp13 genes) were analyzed. Optimal concentrations of primers and probes were variable and defined in a heuristic approach (Table 4). Droplet digital PCR reactions were performed on the QX200 system (Bio-Rad) using a one-step ddPCR Advanced kit according to manufacturer’s instructions (BioRad). Optimal combinations of targets in the single-step procedure were N+RdRP (IP4, nsp12) and E+Helicase (nsp13) as shown in Figure 1 . In brief, 10 pL of RNA was added to 5pL ddPCR Supermix, 1 pL of DTT 300nM and, 2 pL of reverse transcriptase in a final volume of 20 pL. RT and PGR amplification were done in an ICycler PGR instrument (Bio-Rad) according to the following steps: 1 cycle [25°C/3mn, 50°C/60min, 95°C/10min], and 40 cycles [95°/30sec, 55°C (N/IP4) or 59°C (E/nsp13)/1 mn] and a termination step of 98°C/10min. All cycles were performed with a ramping rate of 2°C/sec.

[0170] The limit of detection (LoD) determination was based on the establishment of the limit of blank (LoB). LoB was calculated according to a modified version of the procedure described by Armbruster and Pry (22, 23). The four LoB defined for RdRP (n=3), helicase (n=3), E (n=3), and N (n=4) genes amplification resulted from the analysis of 86 ddPCR replicates containing a mean droplet number of 16188 ±1317 performed on 12 SARS-CoV-2-negative human nasopharyngeal specimens collected during the pre-COVID-19 era. LoD was established for each SARS-CoV-2 target gene analyzed following the same guidelines.

House-keeping genes selection

[0171] In addition, to control the quality of RNA, we tested on clinical samples four different cellular genes: Glyceraldehyde Phosphate Dehydrogenase, GAPDH, Hypoxanthine Phosphoribosyltransferase 1 , HPRT1, RACK1, Receptor for Activated C Kinase 1 , and RPP30, Ribosomal Protein p30. RACK1, the most strongly expressed of the tested set was retained (see Figure 7) (24, 25).

Virus amplification and flow-cytometry

[0172] The strain BetaCoV/France/IDF0372/2020 was supplied by the Pr. S. van der Werf head of the National Reference Centre for Respiratory Viruses hosted by Institut Pasteur (Paris, France). Viral stocks were titrated on Vero E6 cells by plaque assays. [0173] Human A549-ACE2 cells, which have been modified to stably express ACE2 via lentiviral transduction (Pr. Olivier Schwartz, Institut Pasteur, Paris, France). Human Calu-3 (HTB-55) and Vero E6 cells were purchased from ATCC and maintained at 37°C in a humidified atmosphere with 5% CO2. Calu3 and Vero E6 cells were cultured in DMEM (Sigma) supplemented with 10% FBS (Gibco) and 100U/ml penicillinstreptomycin (Thermo Fisher Scientific). The medium of Calu3 was also supplemented with 1 mM sodium pyruvate (Thermo Fisher Scientific) and 10mM HEPES (Thermo Fisher Scientific).

[0174] Cells were fixed in 4% PFA for 15 to 30 minutes at RT and staining was performed in PBS, 1% BSA, 0.05% sodium azide, 0.05% Saponin. Cells were incubated with antibodies recognizing the spike protein of coronaviruses (SARS_Ssd3 293, a kind gift from Dr. Nicolas Escriou, Institut Pasteur, Paris, France), and then with secondary antibodies for 30 minutes at RT (anti-mouse-Alexa Fluor-647). Surface staining was performed before fixation, in PBS 1 % BSA. Cells were incubated with primary antibodies, and then with secondary antibodies for 30 minutes at RT. Cells were fixed for 15 minutes in 4% PFA. Cells were acquired on an Attune NxT Flow Cytometer (Thermo Fisher) and data analyzed with FlowJo software.

Data analysis and statistical tests

[0175] Data were subsequently analyzed with QuantaSoft™ software (version 1.7.4, Bio-Rad). Statistical analyses were performed using Prism 8.4.2 statistical package (GraphPad, San Diego, CA, USA). Numerical variables were summarized by their mean and standard deviation or median and interquartile range according to their types of distribution (normal or not). They were compared either by a Student’s t or by Mann-Whitney U tests as appropriate. Categorical variables were summarized as frequencies that were compared by Fisher’s exact test. All the tests were two-sided and the level of significance was set at P < 0.05. 2-Steps ddPCR

[0176] Reverse transcription was performed using iScript Advanced cDNA Synthesis kit for RT-qPCR with 5-14,8 pL of RNA in a final volume of 20 pL according to manufacturer’s instructions in an iCycler PGR instrument (Bio-Rad) 42°C 30min, 85°C 5min. Droplet digital PGR reactions were performed on the QX200 Droplet Digital PGR system using 5 pL of cDNA and 11 pL of 2X ddPCR Supermix for probes no dllTP (BioRad) in a final volume of 22 pL. PGR amplification was conducted in an iCycler PGR instrument (Bio-Rad), 10min 95°C (ramp rate of 2,5°C/sec), 40 cycles 94°C for 30s (ramp rate of 2,5°C/sec) and 59°C (ramp rate of 2°C/sec) for 1 mn, 10min 98°C (ramp rate of 2,5°C/sec).

Example 2: Pre-ddPCR steps and ddPCR

[0177] We proceeded to “back to back” comparisons of in-house developed ddPCR test with a Bio-Rad kit (Cat#1200802) that targets two different fragments of the SARS-CoV-2 RNA N gene (N1 and N2) and RPP30 mRNA as control. Overall, our results were in agreement with that of the Bio-Rad kit. We noticed, however, that in the very high or low ranges of SARS-CoV-2 RNA concentrations, interpretation of the outcome of the commercial kit can be complexified by the constitutive presence of RPP30-positive droplets representing a third type of amplicon that “geographically” interferes with virus-positive droplets (see figure 8).

Example 3: Sensitivity of ddPCR on different amplicons along

SARS-CoV-2 genome

[0178] Replication of the coronaviruses is characterized by the production beside the full-length genome of a set of sub-genomic RNA (sgRNA) with heterogeneous 5’ but identical 3’ ends. The nine major sgRNA of SARS-CoV-2 are encoded by the last third of the viral genome while ORF1 a-b sequences encoding non-structural proteins (nsps) are present only in full-length genomic viral RNA. In theory, a SARS-CoV-2 detection aiming at RNA segments transcribed both under genomic and sub-genomic forms is predicted to be more sensitive than targeting nsp-encoding genes. In this context, nucleocapsid (N) gene-containing transcripts have been shown to be the most abundant in infected Vero cells due to the most 3’ position of this gene on the SARS-CoV-2 genome (26).

[0179] A549-ACE2 and Calu-3 cells were infected with SARS-CoV-2 virion stocks obtained from Vero cells. Virus replication was assessed by analyzing the intracellular presence of the viral spike protein by flow cytometry at 24h post-infection at MOI 0.3. About 8.0% of A549-ACE2 and 7.0% of Calu-3 cells were infected (Fig. 2AB).

[0180] We then compared ddPCR outcomes on different segments of the viral genome to assess whether this technique is capable to differentiate an active replication process from the presence of genome-loaded viral particles. Two targets were mapping in the 5’ non-structural part of the genome (RdRP-IP4-nsp12 and Helicase-nsp13) while two others were located in structural proteins-encoding genes (envelop E and nucleocapsid N) in the 3’ part of SARS-CoV-2. Four types of RNA samples were tested: RNA extracted from SARS-CoV-2 stocks obtained from a culture on Vero cells, and RNA extracted from A549-ACE2, and Calu-3 exposed to SARS-CoV-2 for 24h at 0.3MOI as previously mentioned. RNA from CaCo-2 cells infected with SARS-CoV-2 for the same time and by the same MOI was also investigated. As expected, we observed that non- structural genes, and particularly N, were consistently detected in larger quantities than nsps-encoding genes (Figure 2A). The situation, presumably due to a large amount of sgRNAs present in replicating host cells, prompted us to privilege the N gene as the principal target for SARS-CoV-2 detection by ddPCR in clinical samples. N gene was associated in a dual-color assay with RdRP-IP4 as its amplification was, in our hands, incompatible with concomitant amplification of E gene amplicon. E gene amplification was, by contrast, compatible with helicase gene (nsp13) amplification.

Example 4: Patients analyzed for SARS-CoV-2 RNA presence

[0181] A series of 208 samples from the respiratory airway (95% were nasopharyngeal swabs) arrived in the frame of the on-call activity of the Cellule d’ Intervention Biologique d’Urgence (CIBU) between the March 14^th and April 13^th, 2020. Patients were recruited in hospitals located in a large area around Paris (Compiegne, Longjumeau, and Orsay). A subset of 13 samples (6.2%) have been scored as clearly positive for SARS-CoV-2 by RT-qPCR while the remaining 195 were either undetermined (n=8, 3.8%) or negative (n=187, 89.9%). The clinical and biological features of patients included are summarized in Table 4. A subset of 16.9% (n=35/208) of the patients tested did not present any respiratory symptoms (such as dyspnea, flulike syndrome, fever, coughing, ARD, or anosmia-ageusia) evocative of COVID-19. Likewise, in a large subset of patients (31%, n=64/207) no notion of the previous contact with a COVID-19 patient was found.

nce for nd probe Pasteur Pasteur Pasteur t al. t al. t al.

[0182] Another series of 44 RNA extracted from different brain territories of 3 patients deceased from a COVID-19-associated encephalitis was also analyzed for presence of SARS-CoV-2 RNA.

Example 5: Sample quality and ddPCR

[0183] We first decided to assess the quality of RNA extracted from clinical specimens as the quality of samples is known to be crucial to obtain a consistent result. To this aim, we had to amplify a cellular gene. SARS-CoV-2 is known, however, to strongly suppress host-cell transcription, a situation that implies to look for highly expressed cellular genes when checking RNA quality of coronavirus-infected cells (26). We, thus, tested respiratory samples for the presence of RACK1 (Receptor for Activated C Kinase 1 ) mRNA that is more highly and stably expressed in airway cells than others well-known candidates (GAPDH, HPRT1, RPP30). Its absence is, thus, a conservative indicator for a lack of adequacy of the biological material. A similar approach was used on brain tissue using HPRT1 (Hypoxanthine Phosphoribosyl Transferase 1 ) mRNA as a target. A small subset of RNA from airway samples (n=10/208, 4.8%), all initially negative for SARS-CoV-2 RNA, was also negative for RACK1 mRNA. This situation implied that in case of a negative result for SARS-CoV-2 amplification on these samples we may neither conclude to presence nor to the absence of viral RNA. These samples were excluded from further analyses. Only two brain samples were negative (n=2/44, 4.5) for HPRT1 mRNA.

Example 6: SARS-CoV-2 RNA detection by ddPCR on clinical samples

[0184] Concerning RNA extracted from respiratory tract specimens, 13 out of 13 (100.0%) initially SARS-CoV-2 RNA positive by RT-qPCR were confirmed, 3 out of 8 (37.5%) undetermined samples were shown to be positive while another one was undetermined due to a low positive droplet number for N gene (n=1 /8, 12.5%). Among the initially negative subset, 8 out of the 177 informative samples (4.5%) were found to carry amounts of SARS-CoV-2 RNA at levels located above the LoD (> 6 droplets/reaction) on both viral targets (N and RdRP-IP4) (Figure 3). Five additional samples (n=5/177, 2.8%) displayed droplets above the LoD for a single viral target (N in four cases, and IP4 in the remaining one) and should, thus, be considered as undetermined according to the diagnostic frame defined by the WHO. Overall, it was between 5.9% (n=11/185) and 9.2% (n=17/185) patients scored as “non-positive” by RT- QPCR who presented a signal for SARS-CoV-2 RNA by ddPCR.

[0185] We observed that mean values of the cycle of quantification (Cq) for RACK1 were significantly different between positive (Cq, mean±SD=31 ,5±3.7) and negative (34.0±3.8) respiratory samples for SARS-CoV-2 RNA (P=0.0024, Figure 3B) indicating, as stressed by others, that sample quality is a decisive parameter to detect the virus (27).

[0186] Concerning SARS-CoV-2 detection in brain tissues, three samples from a single patient were found positive by ddPCR. Positive tissues were the olfactory bulb, olfactory tract, and the middle frontal gyrus.

Example 7: Clinical and biological features of patients with SARS-CoV-2

[0187] We next wonder whether the patients presenting SARS-CoV-2 by ddPCR were different from those who remained negative. Due to the small number of patients concerned and to reach a degree of significance, we decided to examine clinical and biological features both for those patients who gave a frankly positive signal (n=11 patients) and also those who remained undetermined due to the failure to amplify one of the two SARS-CoV-2 RNA targets (n=6). Out of these 17 patients, fifteen recovered (88.2%) but two died (11 .8%) after 5 and 8 days of hospitalization respectively. We observed that a large proportion (59.0%, n=10/17) of the patients positive in ddPCR presented some degree of lymphopenia (<1000 lymphocytes/pL) while 76.4% (n=13/17) displayed increased plasma levels of C-reactive protein (>6mg/L). CT imaging analysis indicated that 63.3% (n=7/11 ) of the patients presented with a lung parenchyma pattern evocative of COVID-19. It was the case for the two deceased patients. [0188] We compared thereafter these 17 patients with the 168 who remained completely negative for SARS-CoV-2 RNA by ddPCR but were informative for cellular mRNA measurement. Subjects positive for ddPCR tended to be younger (57.2±20.2 years vs 66.7±22.0, P=0.0514, ns, Figure 4A). The prevalence of several symptoms was statistically different between those 17 patients and the negatives ones. It was the case of the fever (70.6% vs 13.0%, P=8.0 E-06), the cough (27.7% vs 2.9%, P=0.001 ) and asthenia (27.7% vs 7.3%, P=0.012, Figure 4B). Concerning medical antecedents, no significant difference was detected albeit type 2 diabetes (35.2% vs 15.1 %, P=0.084, ns) tended to be more frequent among patients positive for ddPCR (not shown). Overall, patients positive by ddPCR can be affected by a severe form of COVID-19 and present more often symptoms than SARS-CoV-2 RNA(-) subjects.

[0189] Our results, thus, indicate that improvements in molecular detection of SARS-CoV-2 can be obtained at different steps of the process going from proper sampling, concentration factor between virus stabilization medium, and extracted RNA resuspension, reverse-transcription, and qPCR amplification kits, or the choice of the viral gene target on SARS-CoV-2 RNA. In conclusion, in the diagnostic conditions of France, the use of ddPCR instead of qRT-PCR might slightly increase the rate of positivity for SARS-CoV-2 RNA in upper airways.

Example 8: Discussion

[0190] The issue of SARS-CoV-2 RNA detection in biological samples is of considerable importance and turned-out to be problematic from the onset of the pandemic. In medical practice, it conditions the management, the monitoring, and medical procedures applied to patients with severe respiratory disease. It is also determinant for their safe discharge in the resolution phase. Outside medical institutions, when the disease is benign, a positive test for SARS-CoV-2 RNA implies that the patient has to self-isolate to avoid a further spread of the virus. In Public Health, performant SARS-CoV-2 RNA detection is important to identify clusters of transmission, to monitor properly the circulation of the virus by calculating a realistic reproduction number (R), and to estimate the burden of COVID-19-associated morbidity and fatality. Serological tests have been considered as an efficient surrogate to molecular detection and developed accordingly in the course of the pandemic. It appears currently that the sensitivity of antibody detection is still too low in the initial phase of infection (28). Likewise, the sensitivity of computed tomography once claimed to surpass that of PCR, turned out to be suboptimal with a high rate of false-positive (29).

[0191 ] As a consequence, the problem of the false-negative COVID-19 diagnosis received considerable attention since the onset of the pandemic. The true proportion of false-negative PCR is difficult to estimate. This is primarily due to the fact that the causes of false-negative qPCR are diverse. In most cases, a false negative result is either due to the virus disappearance from the territory sampled or to a merely inadequate sampling rather than to a failure of qPCR to detect viral RNA (27). In case of low biotic burden, the circumstance of choice for the ddPCR use, the amount of biological fluid extracted and the concentration factor from the biological sample to RNA solution ready for amplification tightly related to the extraction method represent also critical parameters (21 , 30, 31 ). Similarly, when viral loads are low, the reduction of the limit of blank and the limit of detection are also paramount to allow a comfortable detection of SARS-CoV- 2 RNA. These parameters are intrinsic to each combination of primers and probes. Regarding probe chemistry, in our hands, dark quenchers such as Black Hole quencher (BHQ) or Iowa Black quencher (IBQ) displayed similar performances in two-steps and single-step reactions respectively.

[0192] Overall, with the present series of samples, we provide a real-world assessment of ddPCR capacity to detect false-negative samples. Sample assayed with ddPCR confirmed 100.0% of samples initially scored positive by qPCR and changed with high confidence the status of around 6% of samples previously considered as nonpositive for SARS-CoV-2 RNA. This latter ratio might be viewed as rather low but should be replaced in the context of the initial phase of the pandemic to which pertains the current series where 17% and 31 % of patients tested neither displayed any symptoms nor any context of the previous contact for COVID-19 patients. A more targeted series might have yielded substantially higher rates of positive PCR. Our work is in agreement with the data of F. Yu and coworkers who observed using ddPCR a rate of 0.9% falsenegative nasal and throat swabs (n=1/112) and 32.0% of positive samples among undetermined samples (n=8/25) (32). In light of our results, we consider that in the situation of the French health system, ddPCR should be applied to those samples considered as “undetermined” by qPCR or to negative nasopharyngeal samples obtained long after symptoms onset (>10 days) in a context of high probability of COVID- 19 substantiated by evocative symptoms, positive chest imagery or previous contact with confirmed cases.

[0193] In addition, we provide in the current work data concerning the quantification of amplification products from structural and non-structural SARS-CoV-2 genes with the aim to estimate the replicative activity of the virus. We consider that our result should be confirmed on a larger series and positioned in a more detailed clinical landscape in order to properly assess the place of Structural/Non-structural genes ratio as a biomarker of replicative infection.

[0194] In conclusion, our work showed in a French context, that ddPCR use might increase from 6% the number of positive cases diagnosed (ie around 1.8 million additional cases in the World on September 16^th 2020). However, due to the limited availability of ddPCR apparatus, its use should be preferably focused on severe COVID- 19 cases with fluctuating viral loads, on undetermined cases with a single positive target on SARS-CoV-2, or before the discharge of convalescent hospitalized patients to ascertain their negativity. In addition, we consider that including the SARS-CoV-2 nucleocapsid (N) sequence, a marker of viral replication, as a primary target in first line diagnosis will increase the sensitivity of PCR tests. REFERENCES

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Claims

1 . An in vitro method for detecting SARS-CoV-2 in a sample, comprising: subjecting at least one subject sample to a reverse transcription reaction to generate a cDNA copy of RNA in the at least one sample; amplifying any resultant cDNA formed using a first forward primer and a first reverse primer that amplify all or part of a first SARS-CoV-2 gene selected from the group consisting of spike (S), envelope (E), membrane (M), nucleocapsid (N), accessory protein (ap) 3a (ap3a), ap3b, ap3c, ap3d, ap6, ap7a, ap7b, ap8, ap9a, ap9b, ap9c, ap10 and ap14, to form a first amplified product; detecting any first amplified product formed using a first probe that hybridizes to the first amplified product; amplifying any resultant cDNA formed using a second forward primer and a second reverse primer that amplify all or part of a second SARS-CoV-2 gene selected from the group consisting of non-structural protein (nsp) 1 (nsp1 ), nsp1 , nsp2, nsp3, nsp4, nsp5, nsp6, nsp7, nsp8, nsp9, nsp10, nsp11 , nsp12, nsp13, nsp14, nsp15 and nsp16, to form a second amplified product; and detecting any second amplified product formed using a second probe that hybridizes to the second amplified product.

2. The method of claim 1 , wherein the first SARS-CoV-2 gene is selected from the group consisting of N, E, and ap7a.

3. The method of claim 1 or 2, wherein the second SARS-CoV-2 gene is selected from the group consisting of nsp12 and nsp13.

4. The method of any one of claims 1 to 3, wherein:

(A) when the first SARS-CoV-2 gene is N the first forward primer hybridizes to the sequence TAGCAGGAGAAGTTCCCC (SEQ ID NO: 14) and the RNA equivalent thereof; the first reverse primer hybridizes to the sequence TTGAGAGCAAAATGTCTG (SEQ ID NO: 15) and the RNA equivalent thereof; and the first probe hybridizes to the sequence AATCTGTCAAGCAGCAGCAA (SEQ ID NO: 16) and the RNA equivalent thereof;

(B) when the first SARS-CoV-2 gene is E the first forward primer hybridizes to the sequence ACGCTATTAACTATTAACGTACCTGA (SEQ ID NO: 20) and the RNA equivalent thereof; the first reverse primer hybridizes to the sequence TGTGTGCGTACTGCTGCAATAT (SEQ ID NO: 21 ) and the RNA equivalent thereof; and the first probe hybridizes to the sequence CGAAGCGCAGTAAGGATGGCTAGTGT (SEQ ID NO: 22) and the RNA equivalent thereof;

(C) when the second SARS-CoV-2 gene is nsp12 the second forward primer hybridizes to the sequence CGAAATCATACCAGTTACC (SEQ ID NO: 17) and the RNA equivalent thereof; the second reverse primer hybridizes to the sequence CCTATATTAACCTTGACCAG (SEQ ID NO: 18) and the RNA equivalent thereof; and the second probe hybridizes to the sequence CCTGGCGTGGTTTGTATGA (SEQ ID NO: 19) and the RNA equivalent thereof; and

(D) when the second SARS-CoV-2 gene is nsp13 the second forward primer hybridizes to the sequence CTGGTTCTAGTGTGCCCTTA (SEQ ID NO: 23) and the RNA equivalent thereof; the second reverse primer hybridizes to the sequence CGTTGTCCTGCTGAAATTGT (SEQ ID NO: 24) and the RNA equivalent thereof; and the second probe hybridizes to the sequence TTCCGAGGAACATGTCTGGACCT (SEQ ID NO: 25) and the RNA equivalent thereof.

5. The method of any one of claims 1 to 3, wherein:

(A) when the first SARS-CoV-2 gene is N the first forward primer comprises the sequence GGGGAACTTCTCCTGCTA (SEQ ID NO: 2) and the RNA equivalent thereof; the first reverse primer comprises the sequence CAGACATTTTGCTCTCAA (SEQ ID NO: 3) and the RNA equivalent thereof; and the first probe comprises the sequence TTGCTGCTGCTTGACAGATT (SEQ ID NO: 4) and the RNA equivalent thereof;

(B) when the first SARS-CoV-2 gene is E the first forward primer comprises the sequence ACAGGTACGTTAATAGTTAATAGCGT (SEQ ID NO: 8) and the RNA equivalent thereof; the first reverse primer comprises the sequence ATATTGCAGCAGTACGCACACA (SEQ ID NO: 9) and the RNA equivalent thereof; and the first probe comprises the sequence ACACTAGCCATCCTTACTGCGCTTCG (SEQ ID NO: 10) and the RNA equivalent thereof;

(C) when the second SARS-CoV-2 gene is nsp12 the second forward primer comprises the sequence GGTAACTGGTATGATTTCG (SEQ ID NO: 5) and the RNA equivalent thereof; the second reverse primer comprises the sequence CTGGTCAAGGTTAATATAGG (SEQ ID NO: 6) and the RNA equivalent thereof; and the second probe comprises the sequence TCATACAAACCACGCCAGG (SEQ ID NO: 7) and the RNA equivalent thereof; and

(D) when the second SARS-CoV-2 gene is nsp13 the second forward primer comprises the sequence TAAGGGCACACTAGAACCAG (SEQ ID NO: 1 1 ) and the RNA equivalent thereof; the second reverse primer comprises the sequence ACAATTTCAGCAGGACAACG (SEQ ID NO: 12) and the RNA equivalent thereof; and the second probe comprises the sequence AGGTCCAGACATGTTCCTCGGAA (SEQ ID NO: 13) and the RNA equivalent thereof.

6. The method of any one of claims 1 to 3, wherein:

(A) when the first SARS-CoV-2 gene is N the first forward primer consists of the sequence GGGGAACTTCTCCTGCTA (SEQ ID NO: 2) and the RNA equivalent thereof; the first reverse primer consists of the sequence CAGACATTTTGCTCTCAA (SEQ ID NO: 3) and the RNA equivalent thereof; and the first probe consists of the sequence TTGCTGCTGCTTGACAGATT (SEQ ID NO: 4) and the RNA equivalent thereof; (B) when the first SARS-CoV-2 gene is E the first forward primer consists of the sequence ACAGGTACGTTAATAGTTAATAGCGT (SEQ ID NO: 8) and the RNA equivalent thereof; the first reverse primer consists of the sequence ATATTGCAGCAGTACGCACACA (SEQ ID NO: 9) and the RNA equivalent thereof; and the first probe consists of the sequence ACACTAGCCATCCTTACTGCGCTTCG (SEQ ID NO: 10) and the RNA equivalent thereof;

(C) when the second SARS-CoV-2 gene is nsp12 the second forward primer consists of the sequence GGTAACTGGTATGATTTCG (SEQ ID NO: 5) and the RNA equivalent thereof; the second reverse primer consists of the sequence CTGGTCAAGGTTAATATAGG (SEQ ID NO: 6) and the RNA equivalent thereof; and the second probe consists of the sequence TCATACAAACCACGCCAGG (SEQ ID NO: 7) and the RNA equivalent thereof; and

(D) when the second SARS-CoV-2 gene is nsp13 the second forward primer consists of the sequence TAAGGGCACACTAGAACCAG (SEQ ID NO: 1 1 ) and the RNA equivalent thereof; the second reverse primer consists of the sequence ACAATTTCAGCAGGACAACG (SEQ ID NO: 12) and the RNA equivalent thereof; and the second probe consists of the sequence AGGTCCAGACATGTTCCTCGGAA (SEQ ID NO: 13) and the RNA equivalent thereof.

7. The method of any one of claims 1 to 6, wherein amplification of the first amplified product using the first forward primer and the first reverse primer is performed in a first container and amplification of the second amplified product using the second forward primer and the second reverse primer is performed in a second container.

8. The method of any one of claims 1 to 7 , further comprising quantifying the RNA transcripts of the first SARS-CoV-2 gene in the at least one sample and/or quantifying the RNA transcripts of the second SARS-CoV-2 gene in the at least one sample.

9. The method of claim 8, further comprising calculating the ratio of the quantified RNA transcripts of the first SARS-CoV-2 gene to the quantified RNA transcripts of the second SARS-CoV-2 gene and comparing the calculated ratio to a threshold value; wherein, when the value of the ratio is higher than a threshold value, the SARS- CoV-2 is replicating in the sample.

10. An in vitro method for characterizing the replicative activity of SARS-CoV- 2 in a sample, comprising: subjecting at least one subject sample to a reverse transcription reaction to generate a cDNA copy of RNA in the at least one sample; amplifying any resultant cDNA formed using a first forward primer and a first reverse primer that amplify all or part of a first SARS-CoV-2 gene selected from the group consisting of spike (S), envelope (E), membrane (M), nucleocapsid (N), accessory protein (ap) 3a (ap3a), ap3b, ap3c, ap3d, ap6, ap7a, ap7b, ap8, ap9a, ap9b, ap9c, ap10 and ap14, to form a first amplified product; detecting any first amplified product formed using a first probe that hybridizes to the first amplified product; amplifying any resultant cDNA formed using a second forward primer and a second reverse primer that amplify all or part of a second SARS-CoV-2 gene selected from the group consisting of non-structural protein (nsp) 1 (nsp1 ), nsp1 , nsp2, nsp3, nsp4, nsp5, nsp6, nsp7, nsp8, nsp9, nsp10, nsp11 , nsp12, nsp13, nsp14, nsp15 and nsp16, to form a second amplified product; detecting any second amplified product formed using a second probe that hybridizes to the second amplified product; quantifying the RNA transcripts of the first SARS-CoV-2 gene in the at least one sample and quantifying the RNA transcripts of the second SARS-CoV-2 gene in the at least one sample; and calculating the ratio of the quantified RNA transcripts of the first SARS-CoV-2 gene to the quantified RNA transcripts of the second SARS-CoV-2 gene and comparing the calculated ratio to a threshold value; wherein, when the value of the ratio is higher than a threshold value, the SARS- CoV-2 is replicating in the sample.

11. The method of claim 10, wherein the first SARS-CoV-2 gene is selected from the group consisting of N, E, and ap7a.

12. The method of claim 10 or 11 , wherein the second SARS-CoV-2 gene is selected from the group consisting of nsp12 and nsp13.

13. The method of any one of claims 10 to 12, wherein:

(B) when the first SARS-CoV-2 gene is E the first forward primer hybridizes to the sequence ACGCTATTAACTATTAACGTACCTGA (SEQ ID NO: 20) and the RNA equivalent thereof; the first reverse primer hybridizes to the sequence TGTGTGCGTACTGCTGCAATAT (SEQ ID NO: 21 ) and the RNA equivalent thereof; and the first probe hybridizes to the sequence CGAAGCGCAGTAAGGATGGCTAGTGT (SEQ ID NO: 22) and the RNA equivalent thereof; (C) when the second SARS-CoV-2 gene is nsp12 the second forward primer hybridizes to the sequence CGAAATCATACCAGTTACC (SEQ ID NO: 17) and the RNA equivalent thereof; the second reverse primer hybridizes to the sequence CCTATATTAACCTTGACCAG (SEQ ID NO: 18) and the RNA equivalent thereof; and the second probe hybridizes to the sequence CCTGGCGTGGTTTGTATGA (SEQ ID NO: 19) and the RNA equivalent thereof; and

14. The method of any one of claims 10 to 12, wherein:

15. The method of any one of claims 10 to 12, wherein:

(A) when the first SARS-CoV-2 gene is N the first forward primer consists of the sequence GGGGAACTTCTCCTGCTA (SEQ ID NO: 2) and the RNA equivalent thereof; the first reverse primer consists of the sequence CAGACATTTTGCTCTCAA (SEQ ID NO: 3) and the RNA equivalent thereof; and the first probe consists of the sequence TTGCTGCTGCTTGACAGATT (SEQ ID NO: 4) and the RNA equivalent thereof;

(B) when the first SARS-CoV-2 gene is E the first forward primer consists of the sequence ACAGGTACGTTAATAGTTAATAGCGT (SEQ ID NO: 8) and the RNA equivalent thereof; the first reverse primer consists of the sequence ATATTGCAGCAGTACGCACACA (SEQ ID NO: 9) and the RNA equivalent thereof; and the first probe consists of the sequence ACACTAGCCATCCTTACTGCGCTTCG (SEQ ID NO: 10) and the RNA equivalent thereof;

(C) when the second SARS-CoV-2 gene is nsp12 the second forward primer consists of the sequence GGTAACTGGTATGATTTCG (SEQ ID NO: 5) and the RNA equivalent thereof; the second reverse primer consists of the sequence CTGGTCAAGGTTAATATAGG (SEQ ID NO: 6) and the RNA equivalent thereof; and the second probe consists of the sequence TCATACAAACCACGCCAGG (SEQ ID NO: 7) and the RNA equivalent thereof; and (D) when the second SARS-CoV-2 gene is nsp13 the second forward primer consists of the sequence TAAGGGCACACTAGAACCAG (SEQ ID NO: 1 1 ) and the RNA equivalent thereof; the second reverse primer consists of the sequence ACAATTTCAGCAGGACAACG (SEQ ID NO: 12) and the RNA equivalent thereof; and the second probe consists of the sequence AGGTCCAGACATGTTCCTCGGAA (SEQ ID NO: 13) and the RNA equivalent thereof.

16. The method of any one of claims 10 to 15, wherein amplification of the first amplified product using the first forward primer and the first reverse primer is performed in a first container and amplification of the second amplified product using the second forward primer and the second reverse primer is performed in a second container.

17. An in vitro method for diagnosing whether a patient who is a SARS-CoV-2 carrier has COVID-19 or is at an elevated risk of developing COVID-19, comprising: characterizing the replicative activity of SARS-CoV-2 in a sample by the method of any one of claims 10 to 16 to determine whether SARS-CoV-2 is replicating in the sample; wherein if SARS-CoV-2 is replicating in the sample the patient is diagnosed with COVID-19 or with an elevated risk of developing COVID-19; and/or wherein if SARS-CoV-2 is not replicating in the sample the patient is not diagnosed with COVID-19 or with an elevated risk of developing COVID-19.

18. An in vitro method for characterizing a SARS-CoV-2 infection in a patient over time, comprising: characterizing the replicative activity of SARS-CoV-2 in a first sample taken from a patient at a first timepoint by the method of any one of claims 10 to 16 to determine whether SARS-CoV-2 is replicating in the first sample at the first timepoint; and characterizing the replicative activity of SARS-CoV-2 in a second sample taken from a patient at a second timepoint by the method of any one of claims 10 to 16 to determine whether SARS-CoV-2 is replicating in the second sample at the second timepoint.

19. The method of claim 18, wherein replication of SARS-CoV-2 is higher in the second sample than the first sample and the COVID-19 status of the patient is worsening.

20. The method of claim 18, wherein replication of SARS-CoV-2 is lower in the second sample than the first sample and the COVID-19 status of the patient is improving.

21 . The method of any one of claims 18 to 20, further comprising administering a COVID-19 treatment to the patient after the first timepoint and before the second timepoint.

22. An in vitro method for characterizing a COVID-19 treatment, comprising: administering a COVID-19 treatment to a patient diagnosed as a SARS-CoV-2 carrier and/or diagnosed with COVID-19; and characterizing the replicative activity of SARS-CoV-2 in a sample taken from a patient by the method of any one of claims 10 to 16 to determine whether SARS-CoV-2 is replicating in the sample.

23. The method of claim 22, wherein if SARS-CoV-2 is replicating in the sample the treatment is determined to not be effective; and/or wherein if SARS-CoV-2 is not replicating in the sample the treatment is determined to be effective.

24. The method of any one of claims 21 to 23, wherein the treatment is a COVID-19 vaccine.

25. The method of any one of claims 21 to 23, wherein the treatment is a nonvaccine agent that reduces or eliminates at least one symptom of COVID-19.

26. An in vitro method for determining whether a patient who is a SARS-CoV- 2 carrier is contagious, comprising: characterizing the replicative activity of SARS-CoV- 2 in a sample by the method of any one of claims 10 to 16 to determine whether SARS- CoV-2 is replicating in the sample; wherein if SARS-CoV-2 is replicating in the sample the patient is diagnosed as contagious; and/or wherein if SARS-CoV-2 is not replicating in the sample the patient is not diagnosed as contagious.

27. The method according to any one of claims 1 to 26, wherein the amplification(s) are performed using a real-time PCR (RT-PCR) or quantitative PCR (qPCR) method.

28. The method according to any one of claims 1 to 26, wherein the amplification(s) are performed using a droplet digital PCR (ddPCR) method.

29. A kit for detecting SARS-CoV-2 in a sample, comprising: a first forward primer and a first reverse primer that amplify all or part of a first SARS-CoV-2 gene selected from the group consisting of spike (S), envelope (E), membrane (M), nucleocapsid (N), accessory protein (ap) 3a (ap3a), ap3b, ap3c, ap3d, ap6, ap7a, ap7b, ap8, ap9a, ap9b, ap9c, ap10 and ap14, to form a first amplified product; a second forward primer and a second reverse primer that amplify all or part of a second SARS-CoV-2 gene selected from the group consisting of non-structural protein (nsp) 1 (nsp1 ), nsp1 , nsp2, nsp3, nsp4, nsp5, nsp6, nsp7, nsp8, nsp9, nsp10, nsp11 , nsp12, nsp13, nsp14, nsp15 and nsp16, to form a second amplified product.

30. The kit of claim 29, further comprising a first probe that hybridizes to the first amplified product and a second probe that hybridizes to the second amplified product.

31. The kit of claim 29 or 30, wherein the first SARS-CoV-2 gene is selected from the group consisting of N, E, and ap7a.

32. The kit of any one of claims 29 to 31 , wherein the second SARS-CoV-2 gene is selected from the group consisting of nsp12 and nsp13.

33. The kit of any one of claims 29 to 32, wherein:

(C) when the second SARS-CoV-2 gene is nsp12 the second forward primer hybridizes to the sequence CGAAATCATACCAGTTACC (SEQ ID NO: 17) and the RNA equivalent thereof; the second reverse primer hybridizes to the sequence CCTATATTAACCTTGACCAG (SEQ ID NO: 18) and the RNA equivalent thereof; and the second probe hybridizes to the sequence CCTGGCGTGGTTTGTATGA (SEQ ID NO: 19) and the RNA equivalent thereof; and (D) when the second SARS-CoV-2 gene is nsp13 the second forward primer hybridizes to the sequence CTGGTTCTAGTGTGCCCTTA (SEQ ID NO: 23) and the RNA equivalent thereof; the second reverse primer hybridizes to the sequence CGTTGTCCTGCTGAAATTGT (SEQ ID NO: 24) and the RNA equivalent thereof; and the second probe hybridizes to the sequence TTCCGAGGAACATGTCTGGACCT (SEQ ID NO: 25) and the RNA equivalent thereof.

34. The kit of any one of claims 29 to 32, wherein:

35. The kit of any one of claims 29 to 32, wherein:

36. The kit any one of claims 29 to 35, further comprising an internal negative control.

37. The kit of any one of claims 29 to 36, further comprising an internal positive control.

38. The kit of any one of claims 29 to 33, further comprising a reverse transcriptase.

39. The kit of any one of claims 29 to 34, further comprising a DNA polymerase.

40. The kit of any one of claims 29 to 39, for use in a method according to any one of claims 1 to 28.