KR102281380B1 - Primers specifically binding to S gene for detecting SARS-CoV-2 and kit comprising the same - Google Patents

Abstract

The present invention relates to a primer for detecting a 2019-novel coronavirus (2019-nCoV or SARS-CoV-2) that specifically binds to the S gene and a kit comprising the same and, more particularly, to a primer set for detecting SARS-CoV-2 that specifically binds to the S gene, a probe, a composition comprising the primer set and the probe, a kit, and a method for detecting SARS-CoV-2 using the kit.

Description

Primers specifically binding to S gene for detecting SARS-CoV-2 and kit comprising the same}

The present invention relates to a primer for detecting the 2019-novel coronavirus (2019-nCoV or SARS-CoV-2) that specifically binds to the S gene and a kit comprising the same, and more particularly, to a kit that specifically binds to the S gene. It relates to a primer set for detecting SARS-CoV-2, a probe, a composition comprising the primer set and the probe, a kit, and a SARS-CoV-2 detection method using the kit.

The 2019-novel coronavirus (2019-nCoV) was first reported on December 31, 2019. The Chinese government reported to the World Health Organization (WHO) that a number of patients developed respiratory pneumonia of unknown cause at the Huanan Fish Market in Wuhan, Hubei Province. The virus, which started in the Huanan fish market, quickly spread beyond community transmission and worldwide through human-to-human transmission. This new virus was announced as a coronavirus by the Wuhan Sanitation Bureau, and its gene was announced on January 07, 2020 to be a new strain of coronavirus that causes respiratory infections, not SARS-Cov or MERS-Cov. The WHO has named it "2019-Novel Coronavirus (2019 nCov)". SARS-Cov (strain B) and MERS-Cov (strain C) belonging to BetaCov cause severe respiratory infectious diseases characterized by acute respiratory symptoms. Mortality rates associated with SARS-Cov and MERS-Cov are up to 10% and 35%, respectively. A genomic sequence isolated from a patient with heterozygous pneumonia in Wuhan in the early stage of COVID-19 was analyzed to have 89% nucleotide homology to bat SARS-like-CoVZXC21 and 82% homology to SARS-CoV, the same betaCoV was found to belong to Its name is SARS-CoV-2.

HCoV is a generally positive-sense, very long (30,000 bp) single-stranded RNA virus. HCoV is composed of structural proteins such as Spike (S), Nucleocapsid (N), Matrix (M) and Envelope (E) and RNA-dependent RNA polymerase (RdRp or It is characterized by two protein groups: nonstructural proteins such as nsp12).

S proteins include S1, S2 and S2' proteins. The S1 protein interacts with host receptors to attach virions to the cell membrane and initiate infection (by their similarity). Binding to human ACE2 and CLEC4M/DC-SIGNR receptors and viral internalization into the endosome of the host cell induce conformational changes of the S glycoprotein. Proteolysis by cathepsin CTSL can reveal the fusion peptide of S2 and activate membrane fusion in the endosome.

The S2 protein mediates the fusion of virions and cell membranes by acting as a class I virus fusion protein. In the current model, proteins have at least three conformations: a pre-fusion native state, a pre-hairpin intermediate state, and a post-fusion state. hairpin state). During viral and target cell membrane fusion, coiled coil regions (7 repeats; heptad repeats) assume a trimer-of-hairpins structure, allowing the fusion peptide to bind to the C-terminal region of the ectodomain. placed close to The formation of this structure appears to lead to the apposition and subsequent fusion of virus and target cell membranes.

The S2' protein acts as a viral fusion peptide that is exposed after S2 cleavage that occurs during viral endocytosis.

The N protein packages the positive-stranded viral genomic RNA into a helical ribonucleocapsid (RNP) and plays a basic role during virion assembly through interaction with the viral genome and membrane protein M. do. It plays an important role in improving the efficiency of subgenomic viral RNA transcription and viral replication.

RdRp is an enzyme important in the life cycle of RNA viruses, including coronaviruses, in which the RdRp active site is highly conserved, representing two consecutive aspartates protruding from the beta-turn structure and cleaving the nucleotide channel. through (free nucleotides can pass through) make them accessible to the surface.

As the COVID-19 pandemic begins and the gene sequence is released, WHO has shared a molecular diagnosis of SARS-CoV-2. For molecular diagnosis, most countries and institutions are using real-time PCR (RT-qPCR), and currently the U.S. Centers for Disease Control and Prevention (CDC) uses 3 A primer-probe set for each species is provided (https://www.cdc.gov/coronavirus/2019-ncov/lab/rt-pcr-panel-primer-probes.html).

However, qRT-PCR assays targeting SARS-CoV-2 have several caveats. First, because of the high similarity between SARS-CoV-2 and SARS-Cov, the primer-probe sets may cross-react, and secondly, the sensitivity of the assay to identify suspicious patients early after hospitalization. that may not be sufficient for In addition, as human-to-human transmission occurs easily and infection is rapidly increasing, the development of a more sensitive and early diagnostic primer-probe set is required.

As a prior art in the related art, an oligonucleotide set capable of detecting the presence of SARS-CoV by distinguishing it from human coronavirus strains HCoV-229E and HCoV-OC43 has been disclosed in US Patent Publication No. US 2010/0279276. .

The present invention has been devised to solve the above problems, and an object of the present invention is to provide a primer set and/or probe capable of specifically detecting SARS-CoV-2.

Another object of the present invention is to provide a composition for detecting SARS-CoV-2 comprising the above-described primer set and probe.

Another object of the present invention is to provide a kit for detecting SARS-CoV-2 comprising the above-described composition for detecting SARS-CoV-2.

Another object of the present invention is to provide a method for detecting SARS-CoV-2 using the above-described kit.

In order to solve the above problems, the present invention provides a primer set for detecting SARS-CoV-2, which specifically binds to the S gene of SARS-CoV-2.

According to a preferred embodiment of the present invention, the primer set can specifically bind to the region of positions 22363 to 22488 of the genome sequence of SARS-CoV-2 (NCBI accession number NC_045512).

According to another preferred embodiment of the present invention, the primer set may include the nucleotide sequences of SEQ ID NOs: 1 and 2.

According to another preferred embodiment of the present invention, the primer set may include a nucleotide sequence having 90% or more sequence identity to the nucleotide sequences of SEQ ID NOs: 1 and 2, respectively.

The present invention also provides a probe for detecting SARS-CoV-2 that specifically binds to the S gene of SARS-CoV-2.

According to a preferred embodiment of the present invention, the probe may include the nucleotide sequence of SEQ ID NO: 3.

According to another preferred embodiment of the present invention, the probe may include a nucleotide sequence having 90% or more sequence identity to the nucleotide sequence of SEQ ID NO: 3.

According to another preferred embodiment of the present invention, each of the probes has one type of fluorescent substance selected from the group consisting of FAM, CAL Fluor Orange 560, VIC, HEX, JOE, Quasar 670 and CY5 at the 5'-end ( fluorophore) may be labeled, and one quencher selected from the group consisting of NFQ-MGB, BHQ-1 BHQ1 nova and BHQ-2 may be labeled at the 3'-end.

Further, the present invention provides a composition for detecting SARS-CoV-2 comprising the above-described primer set and/or probe, and a kit for detecting SARS-CoV-2 comprising the same.

According to a preferred embodiment of the present invention, the compositions and kits include a primer set and a probe that specifically bind to the N gene of SARS-CoV-2; a primer set and probe that specifically binds to the RdRp gene of SARS-CoV-2; or a primer set and probe that specifically binds to the N gene of SARS-CoV-2, and a primer set and probe that specifically binds to the RdRp gene of SARS-CoV-2; may further include.

According to another preferred embodiment of the present invention, the primer set and probe that specifically bind to the N gene bind specifically to the region of positions 29422 to 29514 of the genomic sequence of SARS-CoV-2 (NCBI accession number NC_045512). can do.

According to another preferred embodiment of the present invention, the primer set specifically binding to the N gene includes the nucleotide sequences of SEQ ID NOs: 4 and 5, and the probe specifically binding to the N gene includes the nucleotide sequences of SEQ ID NO: 6 sequence may be included.

According to another preferred embodiment of the present invention, the primer set specifically binding to the N gene includes a nucleotide sequence having at least 90% sequence identity to the nucleotide sequences of SEQ ID NOs: 4 and 5, respectively, and is specific for the N gene The positively binding probe may include a nucleotide sequence having 90% or more sequence identity to the nucleotide sequence of SEQ ID NO: 6.

According to another preferred embodiment of the present invention, the primer set and probe that specifically bind to the RdRp gene are regions of positions 14106 to 14241 of the genome sequence of SARS-CoV-2 (NCBI accession number NC_045512), positions 13963 to It may specifically bind to the region of 14119 or the region of positions 14177 to 14283.

According to another preferred embodiment of the present invention, the primer set specifically binding to the RdRp gene includes a primer set comprising the nucleotide sequences of SEQ ID NOs: 7 and 8; a primer set comprising the nucleotide sequences of SEQ ID NOs: 10 and 11; Alternatively, a primer set comprising the nucleotide sequences of SEQ ID NOs: 13 and 14; and a probe that specifically binds to the RdRp gene may include the nucleotide sequence of SEQ ID NOs: 9, 12 or 15.

According to another preferred embodiment of the present invention, the primer set specifically binding to the RdRp gene comprises a primer set comprising a nucleotide sequence having at least 90% sequence identity to the nucleotide sequences of SEQ ID NOs: 7 and 8, respectively; a primer set comprising a nucleotide sequence each having at least 90% sequence identity to the nucleotide sequences of SEQ ID NOs: 10 and 11; Or a primer set comprising a nucleotide sequence having at least 90% sequence identity to the nucleotide sequences of SEQ ID NOs: 13 and 14, respectively, wherein the probes specifically binding to the RdRp gene include the nucleotides of SEQ ID NOs: 9, 12 or 15 It may comprise a nucleotide sequence having at least 90% sequence identity to the sequence.

According to another preferred embodiment of the present invention, the composition and kit may further include a primer set and a probe that binds to the E gene, which is a common region of SARS-CoV-1 and SARS-CoV-2.

According to another preferred embodiment of the present invention, the primer set binding to the E gene may include the nucleotide sequences of SEQ ID NOs: 16 and 17, and the probe binding to the E gene may include the nucleotide sequence of SEQ ID NO: 18 .

According to another preferred embodiment of the present invention, the composition and kit may further include a primer set and a probe that binds to the human RPP30 (RNase P) gene as an internal control.

According to another preferred embodiment of the present invention, the primer set binding to the human RPP30 gene includes the nucleotide sequences of SEQ ID NOs: 19 and 20, and the probe binding to the human RPP30 gene includes the nucleotide sequence of SEQ ID NO: 21. can

According to another preferred embodiment of the present invention, the composition and kit may further include Taq DNA polymerase.

According to another preferred embodiment of the present invention, the composition and kit can use dUTP and uracil-DNA N-glycosylase (UNG) system for preventing cross-contamination.

Further, the present invention provides a method for detecting SARS-CoV-2 comprising the steps of:

(a) extracting RNA from the isolated biological sample;

(b) amplifying the extracted RNA by performing reverse transcription and polymerase chain reaction (PCR) by treating the above-described kit; and

(c) confirming the result of the PCR amplification with fluorescence.

According to a preferred embodiment of the present invention, the separated biological sample may be any one or more selected from the group consisting of nasopharyngeal or oropharyngeal swab, nasopharyngeal aspirate, bronchoalveolar lavage fluid and sputum. .

According to another preferred embodiment of the present invention, the PCR may be real-time PCR.

According to another preferred embodiment of the present invention, the SARS-CoV-2 detection method may further include (d) confirming the PCR amplification result by measuring a Ct (cycle threshold) value. .

The kit of the present invention has a high detection sensitivity (RdRp gene: 6 copies/rxn, N gene: 2-3 copies/rxn and E gene: 6 copies/rxn) with 31 types including SARS-CoV-1 and MERS-CoV. Only SARS-CoV-2 can be specifically detected without cross-reacting to other viruses and organisms (except that the E gene is a common region of SARS-CoV-1 and SARS-CoV-2, so E primer sets and probes for the gene exhibit cross-reactivity to SARS-CoV-1). In addition, it shows high reproducibility with a CV of less than 3%, and can detect SARS-CoV-2 easily and quickly with a single-step multiplex RT-qPCR in a single tube, and UNG (uracil-DNA N-glycosyl A) system can be used to prevent carry-over contamination.

1 shows the results of performing RT-qPCR with a primer set and probe specific for the S gene using IVT RNA of the S gene of SARS-CoV-2 as a sample (IVT RNA: In-vitro transcribed RNA; NTC : No-template control).
2 shows the results of performing RT-qPCR with a primer set and probe specific for the N gene using IVT RNA of the N gene of SARS-CoV-2 as a sample (IVT RNA: In-vitro transcribed RNA; NTC : No-template control).
3 to 5 show the results of RT-qPCR using three types of primer sets and probes specific for the RdRp gene using IVT RNA of the RdRp gene of SARS-CoV-2 (IVT RNA: In- In vitro transcribed RNA; NTC: No-template control), FIG. 3 shows the results of a primer set and probe comprising the nucleotide sequence of SEQ ID NOs: 7 to 9, and FIG. 4 is a primer comprising the nucleotide sequence of SEQ ID NOs: 10 to 12 Results for the set and probe, and FIG. 5 shows the results for the primer set and probe comprising the nucleotide sequences of SEQ ID NOs: 13 to 15.
6 shows the results of performing RT-qPCR with a primer set and probe for detecting the E gene using IVT RNA of the E gene of SARS-CoV-2 as a sample (IVT RNA: In-vitro transcribed RNA; NTC : No-template control).

As described above, most countries and institutions currently use real-time PCR (RT-qPCR) for molecular diagnosis of SARS-CoV-2, but the high levels of SARS-CoV-2 and SARS-Cov The similarity raises the problem that the primer-probe sets may cross-react and that the sensitivity may not be sufficient to identify suspicious patients early after admission.

Accordingly, the present inventors designed a primer set and probe that specifically bind to the S gene of SARS-CoV-2, and SARS-CoV that has a high sensitivity of 3 copies/rxn and does not show cross-reactivity with SARS-CoV-1. -2 A detection kit was developed and the present invention was completed.

Hereinafter, the present invention will be described in more detail.

A first aspect of the present invention relates to a primer set and/or probe for detecting SARS-CoV-2 that specifically binds to the S gene of SARS-CoV-2.

In the present invention, the term “primer” refers to a polynucleotide capable of serving as an initiation point of nucleic acid synthesis in the template-direction when subjected to conditions in which polynucleotide extension is initiated. Primers can also be used in a variety of other oligonucleotide-mediated synthetic processes, including as initiators of de novo RNA synthesis and in vitro transcription-related processes. Primers are typically single-stranded oligonucleotides (eg, oligodeoxyribonucleotides). The appropriate length of a primer is typically in the range of 6 to 40 nucleotides, more typically in the range of 15 to 35 nucleotides, depending on the intended use. Short primer molecules generally require lower temperatures to form sufficiently stable hybridization complexes with the template. Primers are not required to reflect the exact sequence of the template, but must be sufficiently complementary to hybridize with the template for the primer to be extended. In certain embodiments, the term "primer set" includes a 5'-sense primer that hybridizes complementary to the 5'-end of the nucleic acid sequence to be amplified, and 3'-antisense primers that hybridize to the 3'-end of the sequence to be amplified. It means a set of primers comprising Primers can be labeled, if desired, by incorporating a label that can be detected by spectroscopic, photochemical, biochemical, immunochemical or chemical means. For example, useful labels include: ³² P, fluorescent dyes, electron-dense reagents, enzymes (commonly used in ELISA assays), biotin, or proteins for which haptens and antisera or monoclonal antibodies can be used. .

The primer set for detecting SARS-CoV-2, which specifically binds to the S gene of the present invention, can specifically bind to the region of positions 22363 to 22488 of the genomic sequence of SARS-CoV-2 (NCBI accession number NC_045512). there is.

According to a specific embodiment of the present invention, the primer set for detecting SARS-CoV-2 that specifically binds to the S gene may include the nucleotide sequences of SEQ ID NOs: 1 and 2 as forward and reverse primers, respectively, but is limited thereto doesn't happen For example, the primer set for detecting SARS-CoV-2 that specifically binds to the S gene of the present invention is a nucleotide sequence having 80% or more sequence identity to the nucleotide sequences of SEQ ID NOs: 1 and 2, preferably 85 % or more, more preferably 90% or more, even more preferably 95% or more, most preferably 98% or more sequence identity.

According to another specific embodiment of the present invention, the forward primer of the primer set for detecting SARS-CoV-2 that specifically binds to the S gene may include 19 consecutive nucleotides in the nucleotide sequence of SEQ ID NO: 1. Preferably, it may include 20 or more, 21 or more, 22 or more, 23 or more, 24 or more, or 25 or more consecutive nucleotides in the nucleotide sequence of SEQ ID NO: 1. In addition, the reverse primer may comprise 18 consecutive nucleotides within the nucleotide sequence of SEQ ID NO:2. Preferably, it may include 19 or more, 20 or more, 21 or more, 22 or more, 23 or more, or 24 or more consecutive nucleotides in the nucleotide sequence of SEQ ID NO: 2.

The primer set for detecting SARS-CoV-2 that specifically binds to the S gene of the present invention is used together with a probe that specifically binds to the S gene.

In the present invention, the term "probe" refers to a nucleic acid fragment such as RNA or DNA corresponding to several bases to several hundred bases as short as possible to achieve specific binding with a complementary base sequence, and is labeled, so a specific base The presence or absence of the sequence can be checked. The probe may be manufactured in the form of an oligonucleotide probe, a single stranded DNA probe, a double stranded DNA probe, an RNA probe, or the like. In addition, in order to perform real-time PCR, a fluorescently-labeled probe may be used. More specifically, when using the TaqMan probe, an oligonucleotide labeled with a fluorescent material at the 5' end and a quencher at the 3' end is added to the PCR reaction solution. In addition, real-time PCR using a cycling probe is a highly sensitive detection method composed of a chimeric probe composed of RNA and DNA and RNAse H. Again, the 5' end of the probe is labeled with a fluorescent material and the 3' end of the probe is labeled with a quencher.

The probe is annealed to a specific target sequence located between the forward and reverse primers during the amplification process, and is cleaved by the 5' nuclease activity of Taq polymerase during the extension phase of the PCR cycle to release the fluorescent material (reporter dye). The fluorescence intensity is increased by separation from the quenching material (quenching dye). Fluorescence intensity can be monitored at each PCR cycle by a real-time PCR system.

The SARS-CoV- detection probe of the present invention is labeled with one fluorophore selected from the group consisting of FAM, CAL Fluor Orange 560, VIC, HEX, JOE, Quasar 670 and CY5 at the 5'-end, respectively. and one kind of quenching material selected from the group consisting of NFQ-MGB, BHQ-1 BHQ1 nova and BHQ-2 may be labeled at the 3'-end, but is not limited thereto.

According to a specific embodiment of the present invention, the probe for detecting SARS-CoV-2 that specifically binds to the S gene may include the nucleotide sequence of SEQ ID NO: 3, but is not limited thereto. For example, the probe for detecting SARS-CoV-2 that specifically binds to the S gene of the present invention is a nucleotide sequence having 80% or more sequence identity to the nucleotide sequence of SEQ ID NO: 3, preferably 85% or more, more Preferably it may comprise a nucleotide sequence having at least 90%, even more preferably at least 95%, most preferably at least 98% sequence identity.

According to another specific embodiment of the present invention, the probe for detecting SARS-CoV-2 that specifically binds to the S gene may include 19 consecutive nucleotides in the nucleotide sequence of SEQ ID NO: 3. Preferably, it may include 20 or more, 21 or more, 22 or more, 23 or more, 24 or more, 25 or more consecutive nucleotides.

A second aspect of the present invention relates to a composition and/or kit for detecting SARS-CoV-2 comprising the above-described primer set and/or probe specifically binding to the S gene of SARS-CoV-2.

The composition and kit for detecting SARS-CoV-2 of the present invention may further include the following primer sets and probes:

a primer set and probe that specifically binds to the N gene of SARS-CoV-2;

a primer set and probe that specifically binds to the RdRp gene of SARS-CoV-2; or

A primer set and probe that specifically binds to the N gene of SARS-CoV-2, and a primer set and probe that specifically binds to the RdRp gene of SARS-CoV-2.

In the composition and kit for detecting SARS-CoV-2 of the present invention, the primer set and probe that specifically bind to the N gene are regions of positions 29422 to 29514 of the genomic sequence of SARS-CoV-2 (NCBI accession number NC_045512). can bind specifically to

According to a specific embodiment of the present invention, the primer set for detecting SARS-CoV-2 that specifically binds to the N gene may include the nucleotide sequences of SEQ ID NOs: 4 and 5 as forward and reverse primers, respectively, but is limited thereto doesn't happen For example, the primer set for detecting SARS-CoV-2 that specifically binds to the N gene of the present invention is a nucleotide sequence having 80% or more sequence identity to the nucleotide sequences of SEQ ID NOs: 4 and 5, respectively, preferably 85 % or more, more preferably 90% or more, even more preferably 95% or more, most preferably 98% or more sequence identity.

According to another specific embodiment of the present invention, the forward primer of the primer set for detecting SARS-CoV-2 that specifically binds to the N gene may include 15 consecutive nucleotides in the nucleotide sequence of SEQ ID NO: 4. Preferably, it may include 16 or more, 17 or more, 18 or more, 19 or more, 20 or more, or 21 or more consecutive nucleotides in the nucleotide sequence of SEQ ID NO: 4. In addition, the reverse primer may comprise 15 consecutive nucleotides within the nucleotide sequence of SEQ ID NO:5. Preferably, it may include 16 or more, 17 or more, 18 or more consecutive nucleotides.

According to another specific embodiment of the present invention, the probe for detecting SARS-CoV-2 that specifically binds to the N gene may include the nucleotide sequence of SEQ ID NO: 6, but is not limited thereto. For example, the probe for detecting SARS-CoV-2 that specifically binds to the N gene of the present invention is a nucleotide sequence having 80% or more sequence identity to the nucleotide sequence of SEQ ID NO: 6, preferably 85% or more, more Preferably it may comprise a nucleotide sequence having at least 90%, even more preferably at least 95%, most preferably at least 98% sequence identity.

According to another specific embodiment of the present invention, the probe for detecting SARS-CoV-2 that specifically binds to the N gene may include 18 consecutive nucleotides in the nucleotide sequence of SEQ ID NO: 6. Preferably, it may include 19 or more, 20 or more, 21 or more, 22 or more, 23 or more, 24 or more consecutive nucleotides.

In the composition and kit for detecting SARS-CoV-2 of the present invention, the primer set and probe that specifically bind to the RdRp gene are regions of positions 14106 to 14241 of the genomic sequence of SARS-CoV-2 (NCBI Accession No. NC_045512) , the region at positions 13963 to 14119 or the region at positions 14177 to 14283.

According to a specific embodiment of the present invention, the primer set for detecting SARS-CoV-2 that specifically binds to the RdRp gene may include the nucleotide sequences of SEQ ID NOs: 7 and 8 as forward and reverse primers, respectively, but is limited thereto doesn't happen For example, the primer set for detecting SARS-CoV-2 that specifically binds to the RdRp gene of the present invention is a nucleotide sequence having 80% or more sequence identity to the nucleotide sequences of SEQ ID NOs: 7 and 8, respectively, preferably 85 % or more, more preferably 90% or more, even more preferably 95% or more, most preferably 98% or more sequence identity.

According to another specific embodiment of the present invention, the forward primer of the primer set for detecting SARS-CoV-2 that specifically binds to the RdRp gene may include 15 consecutive nucleotides in the nucleotide sequence of SEQ ID NO: 7. Preferably, it may include 16 or more, 17 or more, 18 or more, 19 or more, 20 or more, or 21 or more consecutive nucleotides in the nucleotide sequence of SEQ ID NO: 1. In addition, the reverse primer may comprise 21 contiguous nucleotides within the nucleotide sequence of SEQ ID NO:8. Preferably, it may include 22 or more, 23 or more, 24 or more, 25 or more, 26 or more, 27 or more consecutive nucleotides.

According to another specific embodiment of the present invention, the primer set for detecting SARS-CoV-2 that specifically binds to the RdRp gene may include the nucleotide sequences of SEQ ID NOs: 10 and 11 as forward and reverse primers, respectively, It is not limited thereto. For example, the primer set for detecting SARS-CoV-2 that specifically binds to the RdRp gene of the present invention is a nucleotide sequence having 80% or more sequence identity to the nucleotide sequences of SEQ ID NOs: 10 and 11, respectively, preferably 85 % or more, more preferably 90% or more, even more preferably 95% or more, most preferably 98% or more sequence identity.

According to another specific embodiment of the present invention, the forward primer of the primer set for detecting SARS-CoV-2 that specifically binds to the RdRp gene may include 18 consecutive nucleotides in the nucleotide sequence of SEQ ID NO: 10. Preferably, it may include 19 or more, 20 or more, 21 or more, 22 or more, 23 or more, or 24 or more consecutive nucleotides in the nucleotide sequence of SEQ ID NO: 10. In addition, the reverse primer may comprise 15 contiguous nucleotides within the nucleotide sequence of SEQ ID NO:11. Preferably, it may include 16 or more, 17 or more, 18 or more, 19 or more, 20 or more, 21 or more consecutive nucleotides.

According to a specific embodiment of the present invention, the primer set for detecting SARS-CoV-2 that specifically binds to the RdRp gene may include the nucleotide sequences of SEQ ID NOs: 13 and 14 as forward and reverse primers, respectively, but is limited thereto doesn't happen For example, the primer set for detecting SARS-CoV-2 that specifically binds to the RdRp gene of the present invention is a nucleotide sequence having 80% or more sequence identity to the nucleotide sequences of SEQ ID NOs: 13 and 14, respectively, preferably 85 % or more, more preferably 90% or more, even more preferably 95% or more, most preferably 98% or more sequence identity.

According to another specific embodiment of the present invention, the forward primer of the primer set for detecting SARS-CoV-2 that specifically binds to the RdRp gene may include 15 consecutive nucleotides in the nucleotide sequence of SEQ ID NO: 13. Preferably, it may include 16 or more, 17 or more, 18 or more consecutive nucleotides in the nucleotide sequence of SEQ ID NO: 13. In addition, the reverse primer may comprise 15 contiguous nucleotides within the nucleotide sequence of SEQ ID NO: 14. Preferably, it may include 16 or more, 17 or more, 18 or more, 19 or more, 20 or more, 21 or more consecutive nucleotides.

According to another specific embodiment of the present invention, the probe for detecting SARS-CoV-2 that specifically binds to the RdRp gene may include the nucleotide sequence of SEQ ID NO: 9, but is not limited thereto. For example, the probe for detecting SARS-CoV-2 that specifically binds to the RdRp gene of the present invention is a nucleotide sequence having 80% or more sequence identity to the nucleotide sequence of SEQ ID NO: 3, preferably 85% or more, more Preferably it may comprise a nucleotide sequence having at least 90%, even more preferably at least 95%, most preferably at least 98% sequence identity.

According to another specific embodiment of the present invention, the probe for detecting SARS-CoV-2 that specifically binds to the RdRp gene may include 21 consecutive nucleotides in the nucleotide sequence of SEQ ID NO: 9. Preferably, it may include 22 or more, 23 or more, 24 or more, 25 or more, 26 or more, 27 or more consecutive nucleotides.

According to another specific embodiment of the present invention, the probe for detecting SARS-CoV-2 that specifically binds to the RdRp gene may include the nucleotide sequence of SEQ ID NO: 12, but is not limited thereto. For example, the probe for detecting SARS-CoV-2 that specifically binds to the RdRp gene of the present invention is a nucleotide sequence having 80% or more sequence identity to the nucleotide sequence of SEQ ID NO: 12, preferably 85% or more, more Preferably it may comprise a nucleotide sequence having at least 90%, even more preferably at least 95%, most preferably at least 98% sequence identity.

According to another specific embodiment of the present invention, the probe for detecting SARS-CoV-2 that specifically binds to the RdRp gene may include 23 consecutive nucleotides in the nucleotide sequence of SEQ ID NO: 12. Preferably, it may include 24 or more, 25 or more, 26 or more, 27 or more, 28 or more, 29 or more consecutive nucleotides.

According to a specific embodiment of the present invention, the probe for detecting SARS-CoV-2 that specifically binds to the RdRp gene may include the nucleotide sequence of SEQ ID NO: 15, but is not limited thereto. For example, the probe for detecting SARS-CoV-2 that specifically binds to the RdRp gene of the present invention is a nucleotide sequence having 80% or more sequence identity to the nucleotide sequence of SEQ ID NO: 15, preferably 85% or more, more Preferably it may comprise a nucleotide sequence having at least 90%, even more preferably at least 95%, most preferably at least 98% sequence identity.

According to another specific embodiment of the present invention, the probe for detecting SARS-CoV-2 that specifically binds to the RdRp gene may include 18 consecutive nucleotides in the nucleotide sequence of SEQ ID NO: 15. Preferably, it may include 19 or more, 20 or more, 21 or more, 22 or more, 23 or more, 24 or more consecutive nucleotides.

In the composition and kit for detecting SARS-CoV-2 of the present invention, a primer set and probe comprising nucleotides of SEQ ID NOs: 7 to 9 (Set 1), primer sets and probes comprising nucleotides of SEQ ID NOs: 10 to 12 ( Set 2) and the primer set and probe (Set 3) containing the nucleotides of SEQ ID NOs: 13 to 15 each showed a similar level of detection limit (LoD) in SARS-CoV-2 detection, and 31 pathogens of Table 9 does not show cross-reactivity to

The composition and kit for detecting SARS-CoV-2 of the present invention may further include a primer set and a probe binding to the E gene, which is a common region between SAR-CoV-1 and SARS-CoV-2.

In the composition and kit for detecting SARS-CoV-2 of the present invention, the primer set and probe binding to the E gene, which is a common region between SAR-CoV-1 and SARS-CoV-2, are selected from the genome sequence of SARS-CoV-2 ( It can specifically bind to the region of positions 26272 to 26382 of NCBI accession number NC_045512).

According to a specific embodiment of the present invention, the primer set for detecting SARS-CoV-2 binding to the E gene may include the nucleotide sequences of SEQ ID NOs: 16 and 17 as forward and reverse primers, respectively, but is not limited thereto. For example, the primer set for detecting SARS-CoV-2 binding to the E gene of the present invention has a nucleotide sequence having 80% or more sequence identity to the nucleotide sequences of SEQ ID NOs: 16 and 17, respectively, preferably 85% or more, More preferably, it may comprise a nucleotide sequence having at least 90% sequence identity, even more preferably at least 95%, and most preferably at least 98% sequence identity.

According to another specific embodiment of the present invention, the forward primer of the primer set for detecting SARS-CoV-2 binding to the E gene may include 20 consecutive nucleotides in the nucleotide sequence of SEQ ID NO: 16. Preferably, it may include 22 or more, 23 or more, 24 or more, 25 or more, 26 or more, or 27 or more consecutive nucleotides in the nucleotide sequence of SEQ ID NO: 16. In addition, the reverse primer may comprise 15 consecutive nucleotides within the nucleotide sequence of SEQ ID NO:17. Preferably, it may include 16 or more, 17 or more, 18 or more, 19 or more, 20 or more, 21 or more consecutive nucleotides.

According to another specific embodiment of the present invention, the probe for detecting SARS-CoV-2 binding to the E gene may include the nucleotide sequence of SEQ ID NO: 18, but is not limited thereto. For example, the probe for detecting SARS-CoV-2 binding to the E gene of the present invention is a nucleotide sequence having 80% or more sequence identity to the nucleotide sequence of SEQ ID NO: 18, preferably 85% or more, more preferably It may comprise a nucleotide sequence having at least 90%, even more preferably at least 95%, most preferably at least 98% sequence identity.

According to another specific embodiment of the present invention, the probe for detecting SARS-CoV-2 binding to the E gene may include 18 consecutive nucleotides in the nucleotide sequence of SEQ ID NO: 18. Preferably, it may include 19 or more, 20 or more, 21 or more, 22 or more, 23 or more, 24 or more consecutive nucleotides.

The composition and kit for detecting SARS-CoV-2 of the present invention may further include a primer set and a probe binding to the human RPP30 (RNase P) gene as an internal control.

According to a specific embodiment of the present invention, the primer set binding to the RPP30 gene may include the nucleotide sequences of SEQ ID NOs: 19 and 20 as forward and reverse primers, respectively, but is not limited thereto. For example, the primer set binding to the RPP30 gene of the present invention is a nucleotide sequence having 80% or more sequence identity to the nucleotide sequences of SEQ ID NOs: 19 and 20, respectively, preferably 85% or more, more preferably 90% or more , even more preferably at least 95%, most preferably at least 98% sequence identity.

According to another specific embodiment of the present invention, the forward primer of the primer set binding to the RPP30 gene may include 15 consecutive nucleotides in the nucleotide sequence of SEQ ID NO: 19. Preferably, it may include 16 or more, 17 or more, 18 or more, 19 or more, 20 or more, or 21 or more consecutive nucleotides in the nucleotide sequence of SEQ ID NO: 10. In addition, the reverse primer may comprise 15 contiguous nucleotides within the nucleotide sequence of SEQ ID NO: 20. Preferably, it may include 16 or more, 17 or more, 18 or more, 19 or more consecutive nucleotides.

According to another specific embodiment of the present invention, the probe binding to the RPP30 gene may include the nucleotide sequence of SEQ ID NO: 21, but is not limited thereto. For example, the probe binding to the RPP30 gene of the present invention is a nucleotide sequence having 80% or more sequence identity to the nucleotide sequence of SEQ ID NO: 21, preferably 85% or more, more preferably 90% or more, even more preferably Preferably, it may comprise a nucleotide sequence having at least 95% sequence identity, most preferably at least 98% sequence identity.

According to another specific embodiment of the present invention, the probe binding to the RPP30 gene may include 17 consecutive nucleotides in the nucleotide sequence of SEQ ID NO: 21. Preferably, it may include 18 or more, 19 or more, 20 or more, 21 or more, 22 or more, 23 or more consecutive nucleotides.

The composition and kit for detecting SARS-CoV-2 of the present invention may also include the following controls:

1) A negative control (No Template Control, NTC) is required to eliminate the possibility of contamination during assay execution and is used for all assays. These NTCs are molecular grade, DNase and RNase-free water.

2) A positive template control is needed to ensure that the assay performed as intended, and is used for all assays starting with the addition of the master mix at a concentration of 300 copies/μL. A positive control consists of a plasmid mixture (RdRp, S, N, E and RNase P).

3) An internal control targeting PNase P (RPP30) is required to confirm the presence of nucleic acids in all samples, and is used for all processed samples.

The composition and kit for detecting SARS-CoV-2 of the present invention may further include one or more other component compositions, solutions or devices suitable for the assay method. Preferably, the diagnostic kit and diagnostic composition may include essential elements necessary for performing a reverse transcription reaction and real-time multiplex PCR. Accordingly, the composition and kit of the present invention, in addition to each primer set specific for the detection gene, a reverse transcriptase for synthesizing a complementary cDNA from RNA, which is a gene of SARS-CoV-2, a DNA polymerase for amplifying the complementary DNA , tube or other suitable container, reaction buffer (pH and magnesium concentrations vary), deoxynucleotides (dNTPs), DNase, RNase inhibitors, DEPC-water, sterile water, and the like.

The kit for detecting SARS-CoV-2 of the present invention may, for example, be configured as shown in Table 1, but is not limited thereto.

Configuration main ingredient sheep 100 tests 1,000 tests 2X One-step RT-qPCR MMX Buffer, dNTP w/dUTP, UNG, reverse transcriptase, RNase inhibitor, Taq DNA polymerase 1,000 μL/tube × 1 10mL/bottle×1 10X Primer/
probe mix - RdRP, S, N genes: SARS-CoV-2 specific
-E gene: SARS-CoV-1/2 common
- RPP30 (RNase P) gene: internal control 200 μL/tube × 1 1 mL/tube × 2 SARS-CoV-2
positive control Mixture of RdRP, S, N, E and RNase P plasmids 50 μL/tube × 1 0.5 mL/tube × 1 nuclease
no additive PCR grade water (water) 500 μL/tube × 1 5mL/bottle×1

The kits of the present invention contain all reagents provided in separate tubes, which are added prior to the assay. Exemplary detection information is shown in Table 2.

fluorescence signal FAM VIC CY5 SARS-CoV-2 specific SARS-CoV-1/2 common internal control (IC) N gene
S gene
RdRp gene E gene RPP30 (RNase P) gene

The kit of the present invention can use dUTP and uracil-DNA N-glycosylase (UNG) system for preventing carryover contamination.

Since PCR is a very sensitive detection method, a false positive may occur due to cross-contamination of the PCR amplification product previously performed. If the dUTP and uracil-DNA N-glycosylase (UNG) system are used, false-positive reactions caused by carryover can be prevented, thereby increasing the reliability of the test. Specifically, the uracil-DNA N-glycosylase (UNG) system uses a dNTP mixture containing dUTP instead of dTTP as a substrate to produce an amplification product containing uracil instead of thymine. Even if the amplification product is cross-contaminated, if the PCR reaction is performed after treatment with uracil-N-glycosylase (UNG) before the next reaction, the contaminated amplification product is degraded and only DNA derived from the sample that does not contain uracil remains the template for PCR reactions. Therefore, cross-contamination by repeated PCR can be prevented.

The kit of the present invention can be used for in vitro diagnostic (In-Vitro Diagnostic, IVD). Therefore, the kit for detecting SARS-CoV-2 of the present invention can be applied to the diagnosis of pneumonia caused by SARS-CoV-2 infection.

A third aspect of the present invention relates to a method for detecting SARS-CoV-2 comprising the steps of:

(a) extracting RNA from the isolated biological sample;

(b) treating the extracted RNA with the kit of the present invention to perform reverse transcription and PCR (polymerase chain reaction) to amplify; and

(c) confirming the result of the PCR amplification with fluorescence.

The kit used for the SARS-CoV-2 detection method of the present invention may include primer sets and probes targeting S, N, and RdRp genes of SARS-CoV-2, respectively, in various combinations.

For example, a primer set and probe targeting each gene may be included alone, or a primer set and probe targeting two or three genes among S, N and RdRp genes may be included. As confirmed in Tables 8 and 9, the primer sets and probes for each gene were 2 to 6 copies/rxn of LoD (RdRp gene: 6 copies/rxn, N gene: 2 copies/rxn and E gene: 6 copies/ rxn), which can detect SARS-CoV-2 with high sensitivity, and crosses against 31 other viruses and organisms, including SARS-CoV-1 and MERS-CoV, as shown in Table 12. Only SARS-CoV-2 can be specifically detected without a reaction.

Therefore, even if the primer sets and probes for each gene are used alone, SARS-CoV-2 can be detected with high sensitivity and without cross-reaction against other viruses and organisms, and the primer sets for three genes and When the probes are used in various combinations, the accuracy of detecting SARS-CoV-2 or diagnosing SARS-CoV-2 infection can be improved.

In the SARS-CoV-2 detection method of the present invention, the separated biological sample is any one selected from the group consisting of nasopharyngeal or oropharyngeal swab, nasopharyngeal aspirate, bronchoalveolar lavage fluid and sputum. It may be abnormal, but any biological sample capable of containing RNA of SARS-CoV-2 isolated from a sample for detecting SARS-CoV-2 or an individual for diagnosing whether or not infected with SARS-CoV-2 may be used without limitation. .

In addition, the subject to be diagnosed with SARS-CoV-2 infection may be a vertebrate, more preferably a human subject.

The SARS-CoV-2 detection method of the present invention may be performed by real-time PCR.

The "real-time PCR" can monitor the process in real time when performing PCR. Thus, data is collected throughout the PCR process, not at the end of the PCR. In real-time PCR, the reaction is characterized by the time point during the cycle when amplification is first detected rather than the amount of target accumulated after a fixed number of cycles. Two methods are mainly used to perform quantitative PCR: dye-based detection and probe-based detection.

The SARS-CoV-2 detection method of the present invention may further include (d) confirming the PCR amplification result by measuring a cycle threshold (Ct) value.

The cycle threshold (Ct) value means the number of cycles in which fluorescence generated in the reaction exceeds a threshold, which is inversely proportional to the logarithm of the initial copy number. Therefore, the Ct value assigned to a particular well reflects the number of cycles in which a sufficient number of amplicons in the reaction have accumulated. The Ct value is the cycle in which an increase in ΔRn was first detected. Rn denotes the magnitude of the fluorescence signal generated during PCR at each time point, and ΔRn denotes the fluorescence emission intensity of the reporter dye (normalized reporter signal) divided by the fluorescence emission intensity of the reference dye. The Ct value is also called Cp (crossing point) in LightCycler. The Ct value represents the point at which the system begins to detect an increase in fluorescence signal associated with exponential growth of the PCR product in a log-linear phase. This period provides the most useful information about the reaction. The slope of the log-linear step represents the amplification efficiency (Eff) (http://www.appliedbiosystems.co.kr/).

On the other hand, TaqMan probes typically contain a primer (e.g., 20-) comprising a fluorophore at the 5'-end and a quencher (e.g., TAMRA or non-fluorescence quencher (NFQ)) at the 3'-end. oligonucleotides longer than 30 nucleotides). The excited fluorescent material transfers energy to a nearby quenching material rather than fluorescing it (FRET = Frster or fluorescence resonance energy transfer; Chen, X., et al., Proc Natl Acad Sci USA, 94(20): 10756- 61 (1997)). Therefore, when the probe is normal, no fluorescence is generated. TaqMan probes are designed to anneal to the internal sites of PCR products.

The TaqMan probe specifically hybridizes to the template DNA during the annealing step, but fluorescence is suppressed by the quencher on the probe. During the extension reaction, the TaqMan probe hybridized to the template is decomposed by the 5' to 3' nuclease activity of Taq DNA polymerase, and the fluorescent dye is released from the probe, the inhibition by the quencher is released, and fluorescence is displayed. In this case, the 5'-end of the TaqMan probe should be located downstream of the 3'-end of the extension primer. That is, when the 3'-end of the extension primer is extended by a template-dependent nucleic acid polymerase, the 5'-end of the TaqMan probe is cleaved by the 5' to 3' nuclease activity of this polymerase, thereby forming a reporter molecule. A fluorescence signal is generated.

The reporter molecule and quencher molecule bound to the TaqMan probe include a fluorescent material and a non-fluorescent material. Fluorescent reporter molecules and quencher molecules that can be used in the present invention can be any known in the art, examples of which are as follows (numbers in parentheses are the maximum emission wavelength in nanometers): Cy2 ^TM (506), YOPRO ^TM -1 (509), YOYO ^TM -1 (509), Calcein (517), FITC (518), FluorX ^TM (519), Alexa ^TM (520), Rhodamine 110 (520), 5-FAM (522), Oregon Green ^TM 500 (522), Oregon Green ^TM 488 (524), RiboGreen ^TM (525), Rhodamine Green ^TM (527), Rhodamine 123 (529), Magnesium Green ^TM (531), Calcium Green ^TM (533), TO-PRO ^TM -1 (533), TOTO1 (533), JOE (548), BODIPY530/550 (550), Dil (565), BODIPY TMR (568), BODIPY558/568 (568) , BODIPY564/570 (570), Cy3 ^™ (570), Alexa ^™ 546 (570), TRITC (572), Magnesium Orange ^™ (575), Phycoerythrin R&B (575), Rhodamine Phalloidin (575), Calcium Orange ^™ (576) ), Pyronin Y (580), Rhodamine B (580), TAMRA (582), Rhodamine Red ^™ (590), Cy3.5 ^™ (596), ROX (608), Calcium Crimson ^™ (615), Alexa ^™ 594 ( 615), Texas Red (615), Nile Red (628), YO-PRO ^TM -3 (631), YOYO ^TM -3 (631), R-phycocyanin (642), CPhycocyanin (648), TO-PRO ^TM - 3 (660), TOTO3 (660), DiD DilC (5) (665), Cy5 ^TM (670), Thiadicarbocyanine (671), Cy5.5 (694), HEX (556), TET (536), VIC (546), BHQ-1 ( 534), BHQ-2 (579), BHQ-3 (672), Biosearch Blue (447), CAL Fluor Gold 540 (544), CAL Fluor Orange 560 (559), CAL Fluor Red 590 (591), CAL Fluor Red 610 (610), CAL Fluor Red 635 (637), FAM (520), Fluorescein (520), Fluorescein-C3 (520), Pulsar 650 (566), Quasar 570 (667), Quasar 670 (705) and Quasar 705 (610). The number in parentheses is the maximum emission wavelength expressed in nanometers.

Suitable reporter-quencher pairs are disclosed in many literatures: Pesce et al., editors, FLUORESCENCE SPECTROSCOPY (Marcel Dekker, New York, 1971); White et al., FLUORESCENCE ANALYSIS: A PRACTICAL APPROACH (Marcel Dekker, New York, 1970); Berlman, HANDBOOK OF FLUORESCENCE SPECTRA OF AROMATIC MOLECULES, 2 ^nd EDITION (Academic Press, New York, 1971); Griffiths, COLOR AND CONSTITUTION OF ORGANIC MOLECULES (Academic Press, New York, 1976); Bishop, editor, INDICATORS (Pergamon Press, Oxford, 1972); Haugland, HANDBOOK OF FLUORESCENT PROBES AND RESEARCH CHEMICALS (Molecular Probes, Eugene, 1992); Pringsheim, FLUORESCENCE AND PHOSPHORESCENCE (Interscience Publishers, New York, 1949); Haugland, RP, HANDBOOK OF FLUORESCENT PROBES AND RESEARCH CHEMICALS, Sixth Edition, Molecular Probes, Eugene, Oreg., 1996; US Pat. Nos. 3,996,345 and 4,351,760.

In addition, the non-fluorescent material used for the reporter molecule and the quencher molecule bound to the TaqMan probe may include a minor groove binding (MGB) moiety. As used herein, the term "TaqMan MGB-conjugate probe" refers to a TaqMan probe conjugated with MGB at the 3'-end of the probe.

MGB is a substance that binds to the minor groove of DNA with high affinity, netropsin, distamycin, lexitropsin, mithramycin, chromomycin A3, olivo olivomycin, anthramycin, sibiromycin, pentamidine, stilbamidine, berenil, CC-1065, Hoechst 33258, DAPI (4-6 diamidino-2-phenylindole), dimers, trimers, tetramers and pentamers of CDPI, N-methylpyrrole-4-carbox-2-amide (MPC) and dimers, trimers, tetramers and pentamers thereof. it's not going to be

Conjugation of the probe and MGB significantly increases the stability of the hybrid formed between the probe and its target. More specifically, increased stability (ie, increased degree of hybridization) results in an increased melting temperature (Tm) of the hybrid duplex formed by the MGB-conjugated probe compared to the normal probe. Therefore, MGB stabilizes the van der Waals force to increase the melting temperature (Tm) of the MGB-conjugated probe without increasing the probe length, resulting in shorter probes (e.g., 21 nucleotides or less) in Taqman real-time PCR under more stringent conditions. makes the use of

In addition, the MGB-conjugated probe removes the background fluorescence more efficiently. Accordingly, the probe of the present invention may be in the form of a TaqMan MGB-conjugate, wherein the length of the probe includes, but is not limited to, 15-21 nucleotides.

In the SARS-CoV-2 detection method of the present invention, exemplary interpretations of results for the positive control and the negative control are shown in Table 3 below.

FAM VIC CY5 decision positive control 25 ≤ Ct ≤ 31 25 ≤ Ct ≤ 31 27 ≤ Ct ≤ 33 available negative control not detected not detected not detected invalidity

In the SARS-CoV-2 detection method of the present invention, exemplary interpretations of results according to cut-off Ct values for each gene are shown in Tables 4 and 5.

Cut-off Ct values for each target in clinical samples Determination of valid results Cut-off Ct value decision Cut-off Ct value RdRp gene
N gene
S gene
E gene ≤40 positivity >40 or N/A voice

case 1 case 2 case 3 case 4 IC (CY5) +/- +/- + - E gene +/- + - - N+S+RdRP gene + - - - Interpretation of results positivity
(SARS-CoV-2 detection) negative (SARS-CoV-2 non-detection,
Sarbecovirus detection) voice invalidity

The SARS-CoV-2 detection method using the kit of the present invention does not show cross-reactivity against 31 pathogens in Table 6. However, since the E gene is a common region of SARS-CoV-1 and SARS-CoV-2, the primer set and probe for the E gene are exceptionally cross-reactive to SARS-CoV-1.

Other high-priority pathogens from the same gene family High priority organisms in the circulating area Human coronavirus 229E Human Adenovirus 1 Human coronavirus OC43 Human Adenovirus 3 Human coronavirus HKU1 Human Cytomegalovirus Human coronavirus NL63 Human rhinovirus B14 SARS-coronavirus Human parainfluenza virus 1 MERS-coronavirus Human parainfluenza virus 2 Human parainfluenza virus 3 Influenza A virus (H1N1) Influenza A virus (H3N2) Influenza B virus Human respiratory syncytial virus A Human respiratory syncytial virus B Human Rhinovirus A1 Coxsackie virus (B3) Echovirus (E6) Mumps virus (H) Measles virus Streptococcus pyogenes Klebsiella pneumoniae Pseudomonas aeruginosa Haemophilus influenzae Streptococcus pneumoniae Legionella pneumophila Mycobacterium Tuberculosis Mycobacterium Avium

Hereinafter, the present invention will be described in more detail by way of Examples. These examples are merely for illustrating the present invention in more detail, and thereby it will be apparent to those skilled in the art that the technical scope of the present invention is not limited to these examples.

Preparation of primers and probes

Primers and probes targeting the S, N and RdRp genes that specifically detect SARS-CoV-2, primers and probes targeting the E gene common to SARS-CoV-1/2, and RPP30 for use as internal controls OligoAnalyzer Tool (https://sg.idtdna.com/calc/analyzer) program and Multiple primer analyzer (https://www.thermofisher.com/kr/) to design primers and probes targeting the (RNase P) gene en/home/brands/thermo-scientific/molecular-biology/molecular-biology-learning-center/molecular-biology-resource-library/thermo-scientific-web-tools/multiple-primer-analyzer.html) Size, Tm value, primer GC content, whether there is a self-complementary sequence in the primer, the possibility of forming a secondary structure, the possibility of homo-dimer and hetero-dimer formation between primers After exclusion, the primers were designed as shown in Table 7.

The designed primer was confirmed that the sequence was specific to SARS-CoV-2 to be detected through BLAST analysis (Nucleotide BLAST, https://blast.ncbi.nlm.nih.gov/Blast.cgi), and Table 6 It was confirmed that there was no possibility of cross-reacting to sequences in the NCBI database including pathogens of However, since the E gene is a common region of SARS-CoV-1 and SARS-CoV-2, the primer set and probe for the E gene were exceptionally BLAST results for SARS-CoV-1.

subset target area Primer/Probe sequence (5'-3') SEQ ID NO: Ref. Regions in the dielectric (NC_045512) SARS-CoV-2 specific primers S S_F1 GGG TTA TCT TCA ACC TAG GAC TTT TC One 22363-22488 S_R1 TCT ACA GTG AAG GAT TTC AAC GTA C 2 S_FAM1 CTG TGC ACT TGA CCC TCT CTC AGA AA 3 N N_F2 GCA GAG ACA GAA GAA ACA GCA A 4 29422-29514 N_R2 GCA CTG CTC ATG GAT TGT T 5 N_FAM2 CTT CCT GCT GCA GAT TTG GAT GAT T 6 RdRp
(Set 1) RdRp_F1 CAT ACA AAC CAC GCC AGG TAG T 7 14106-14241 RdRp_R1 CTT AAT GTA AGG CTT TGT TAA GTC AGT G 8 RdRp_FAM1 ATT AAC CTT GAC CAG GGC TTT AAC TGC A 9 RdRp
(Set 2) RdRp_F2 CAA ACA TAC AAC GTG TTG TAG CTT G 10 13963-14119 RdRp_R2 AGT TGT GGC ATC TCC TGA TGA G 11 RdRp_FAM2 TAA TGA GTG TGC TCA AGT ATT GAG TGA AAT 12 RdRp
(Set 3)
RdRp_F4 CCT TGA CCA GGG CTT TAA C 13 14177-14283 RdRp_R4 TTT TAA CCT CTC TTC CGT GAA G 14 RdRp_FAM4 TGT AAG GCT TTG TTA AGT CAG TGT C 15 SARS-CoV-1/2
common primer E E_F1 GGT ACG TTA ATA GTT AAT AGC GTA CTT C 16 26272-26382 E_R1 AAT ATT GCA GCA GTA CGC ACA C 17 E_CFO560 ACT AGC CAT CCT TAC TGC GCT TCG A 18 internal control RPP30
(RNase P) RPP30 F2 GAT GCA AAT CTG CAA AGG AAA GA 19 RPP30 R2 CCC AAA CAG CAA GCC TAG AT 20 RPP30 QS670_2 TAA GAG GGC CAT ATG ACG TGG CAA 21

SARS-CoV-2 detection

2-1. Sample preparation

As quantified viral isolates of 2019-nCoV are currently not available, tests were performed with in vitro transcribed RNA (IVT RNA) from each gene plasmid (Ref. genome: NC_045512). IVT RNA of S gene (SARS-CoV-2 reference genome: 21536-23500 nt region of NC_045512), IVT RNA of N gene (SARS-CoV-2 reference genome: 28274-29533 nt region of NC_045512), RdRp gene IVT RNA The test was performed with (SARS-CoV-2 reference genome: 13901-15530 nt region of NC_045512) and E gene IVT RNA (SARS-CoV-2 reference genome: 26245-26472 nt region of NC_045512).

2-2. SARS-CoV-2 detection

^{10-fold serial dilutions (10 4} , 10 ³ , 10 ² and 10 ¹ ) of characterized stocks of SARS-CoV-2 synthetic RNA (IVT RNA) of each gene were tested. Specific qPCR experimental conditions are shown in Tables 8 and 9 below.

SARS-CoV-2 Reaction Master Mix ingredient Volume per test (μL) Nuclease-free water 3 2X One-step RT-qPCR MMX 10 10X Primer/Probe Mix 2 sample 5 gun 20

cycling parameters step cycle temperature time data collection UNG culture One One 25℃ 2 minutes - reverse transcription 2 One 50℃ 15 minutes early metamorphosis 3 One 95℃ 5 minutes denaturalization 4 45 95℃ 10 seconds - Annealing 5* 60℃ 30 seconds FAM/VIC/CY5 extension 6 72℃ 10 seconds -

* Data collection in step 5. Fluorescence was detected at 60°C.

1 to 6 , each target gene could be detected in all samples using the primer sets and probes of Table 7 above.

2-3. Check limit of detection (LoD)

To confirm LoD, 10-fold serial dilutions of characterized stocks of SARS-CoV-2 synthetic RNA (IVT RNA) of each gene were added to 5 diluted samples (8 copies/rxn) at concentrations close to LoD. , 6 copies/rxn, 4 copies/rxn, 3 copies/rxn, and 2 copies/rxn), and 20 replicate tests were performed for each of these samples. The LoDs in AB 7500 Fast (Applied Biosystems 7500 Fast) and Bio-rad CFX96 are shown in Tables 10 and 11, respectively.

Check limit of detection on AB 7500 Fast target RNA concentration* Positive/All % repeat detection
(Replicate Detection) Average Ct** standard deviation (Ct) 2019-nCoV_N 8 copies/reaction 24/24 100.0 33.8 0.6 6 copies/reaction 24/24 100.0 34.3 0.5 4 copies/reaction 24/24 100.0 34.9 0.7 3 copies/reaction 23/24 95.8 35.5 0.7 2 copies/reaction 18/24 75.0 36.6 1.2 2019-nCoV_S 8 copies/reaction 24/24 100.0 34.9 0.7 6 copies/reaction 24/24 100.0 35.4 0.7 4 copies/reaction 24/24 100.0 35.9 0.8 3 copies/reaction 24/24 100.0 36.3 0.9 2 copies/reaction 21/24 87.5 37.4 2.0 2019-nCoV_RdRp
(Set 1) 8 copies/reaction 24/24 100.0 34.1 0.5 6 copies/reaction 24/24 100.0 34.3 1.1 4 copies/reaction 22/24 91.7 36.3 1.0 3 copies/reaction 22/24 91.7 36.6 1.1 2 copies/reaction 16/24 66.7 37.2 1.4 2019-nCoV_E 8 copies/reaction 24/24 100.0 32.8 1.1 6 copies/reaction 24/24 100.0 33.1 0.8 4 copies/reaction 18/24 75.0 34.8 1.6 3 copies/reaction 11/24 45.8 35.2 1.7 2 copies/reaction 9/24 37.5 35.3 1.7

Check limit of detection on Bio-rad CFX96 target RNA concentration* Positive/All % repeat detection
(Replicate Detection) Average Ct** standard deviation (Ct) 2019-nCoV_N 8 copies/reaction 24/24 100.0 34.0 0.3 6 copies/reaction 24/24 100.0 34.6 0.3 4 copies/reaction 24/24 100.0 34.9 0.0 3 copies/reaction 24/24 100.0 35.2 0.2 2 copies/reaction 23/24 95.8 36.3 0.3 2019-nCoV_S
(Set 1) 8 copies/reaction 24/24 100.0 34.5 0.1 6 copies/reaction 24/24 100.0 35.1 0.1 4 copies/reaction 24/24 100.0 35.3 0.1 3 copies/reaction 24/24 100.0 35.6 0.1 2 copies/reaction 20/24 83.3 36.5 0.2 2019-nCoV_RdRp 8 copies/reaction 24/24 100.0 34.5 0.4 6 copies/reaction 24/24 100.0 34.8 0.5 4 copies/reaction 24/24 100.0 35.7 0.8 3 copies/reaction 23/24 95.8 36.4 0.6 2 copies/reaction 22/24 91.7 37.5 0.7 2019-nCoV_E 8 copies/reaction 24/24 100.0 32.9 0.2 6 copies/reaction 24/24 100.0 33.4 0.1 4 copies/reaction 21/24 87.5 34.0 0.3 3 copies/reaction 21/24 87.5 35.5 0.1 2 copies/reaction 14/24 58.3 35.8 0.7

* Concentration is expressed as RNA copies/reaction.

* Mean Ct includes only positive results.

As confirmed in Tables 10 and 11, the SARS-CoV-2 detection method using the kit of the present invention exhibits a detection limit (LoD) of 2-6 copies/rxn. Specifically, AB 7500 Fast showed 3-6 copies/rxn and Bio-Rad CFX96 showed 2-6 copies/rxn.

Cross-reaction check

Cross-reactivity against 31 viruses and organisms in Table 6 was tested (wet-tested) with primer sets and probes for each gene in Table 7. All viruses and organisms were tested for nucleic acids, and each virus and organism was spiked directly into the RT-qPCR reaction master mixture of Table 8 at the concentrations shown in Table 12.

cross-reactivity test name supplier density FAM
(RdRp, S and N genes) VIC
(E gene) Human Coronavirus 229E KBPV (KBPV-VR-9) 2x10 ⁴ copies/rxn 0/3 0/3 Human Coronavirus HKU1 ATCC (VR-3262SD) 2x10 ⁴ copies/rxn 0/3 0/3 Human Coronavirus OC43 ATCC (VR-1558DQ) 2x10 ⁴ copies/rxn 0/3 0/3 Human Coronavirus NL63 ATCC (VR-3263SD) 2x10 ⁴ copies/rxn 0/3 0/3 MERS-CoV ATCC (VR-3248SD) 2x10 ⁴ copies/rxn 0/3 0/3 SARS-CoV plasmid 6x10 ³ copies/rxn 0/3 3/3 Human Adenovirus 1 KBPV (VR-1) 2x10 ⁴ copies/rxn 0/3 0/3 Human Adenovirus 3 KBPV (VR-2) 2x10 ⁴ copies/rxn 0/3 0/3 Human Cytomegalovirus KBPV (VR-7) 2x10 ⁴ copies/rxn 0/3 0/3 Human rhinovirus B14 KBPV (VR-39) 2x10 ⁴ copies/rxn 0/3 0/3 Human parainfluenza virus 1 KBPV (VR-44) 2x10 ⁴ copies/rxn 0/3 0/3 Human parainfluenza virus 2 KBPV (VR-45) 2x10 ⁴ copies/rxn 0/3 0/3 Human parainfluenza virus 3 KBPV (VR-46) 2x10 ⁴ copies/rxn 0/3 0/3 Influenza A virus (H1N1) NCCP (42004) 2x10 ⁴ copies/rxn 0/3 0/3 Influenza A virus (H3N2) NCCP (42471) 2x10 ⁴ copies/rxn 0/3 0/3 Influenza B virus NCCP (43027) 2x10 ⁴ copies/rxn 0/3 0/3 Human respiratory syncytial virus A NCCP (43238) 2x10 ⁴ copies/rxn 0/3 0/3 Human respiratory syncytial virus B NCCP (43239) 2x10 ⁴ copies/rxn 0/3 0/3 Human Rhinovirus A1 NCCP (43226) 2x10 ⁴ copies/rxn 0/3 0/3 Coxsackie (B3) NCCP (43221) 2x10 ⁴ copies/rxn 0/3 0/3 Echovirus (E6) NCCP (41001) 2x10 ⁴ copies/rxn 0/3 0/3 Mumps virus (H) NCCP (40001) 2x10 ⁴ copies/rxn 0/3 0/3 Measles virus NCCP (40204) 2x10 ⁴ copies/rxn 0/3 0/3 Streptococcus pyogenes NCCP (72067) 2x10 ⁴ copies/rxn 0/3 0/3 Klebsiella pneumoniae NCCP (72008) 2x10 ⁴ copies/rxn 0/3 0/3 Pseudomonas aeruginosa NCCP (72006) 2x10 ⁴ copies/rxn 0/3 0/3 Haemophilus influenzae NCCP (72020) 2x10 ⁴ copies/rxn 0/3 0/3 Streptococcus pneumoniae NCCP (72003) 2x10 ⁴ copies/rxn 0/3 0/3 Legionella pneumophila NCCP (72002) 2x10 ⁴ copies/rxn 0/3 0/3 Mycobacterium Tuberculosis vircell (MBC034) 1x10 ⁴ copies/rxn 0/3 0/3 Mycobacterium Avium vircell (MBC086) 1x10 ⁴ copies/rxn 0/3 0/3

As confirmed in Table 12, the SARS-CoV-2 detection method using the primer sets and probes targeting each gene of Table 7 did not show cross-reactivity against the 31 pathogens of Table 6. However, since the E gene is a common region of SARS-CoV-1 and SARS-CoV-2, the primer set and probe for the E gene were exceptionally cross-reactive to SARS-CoV-1.

SEQUENCE LISTING <110> GeneCast Co., Ltd. <120> Primers specifically binding to S gene for detecting SARS-CoV-2 and kit comprising the same <130> 1067531 <160> 21 <170> PatentIn version 3.2 <210> 1 <211> 26 <212> DNA <213> <220> <223> S_F1 primer <400> 1 gggttatctt caacctagga cttttc 26 <210> 2 <211> 25 <212> DNA <213> <220> <223> S_R1 primer <400> 2 tctacagtga aggatttcaa cgtac 25 <210> 3 <211> 26 <212> DNA <213> <220> <223> S_FAM1 probe <400> 3 ctgtgcactt gaccctctct cagaaa 26 <210> 4 <211> 22 <212> DNA <213> <220> <223> N_F2 primer <400> 4 gcagagacag aagaaacagc aa 22 <210> 5 <211> 19 <212> DNA <213> <220> <223> N_R2 primer <400> 5 gcactgctca tggattgtt 19 <210> 6 <211> 25 <212> DNA <213> <220> <223> N_FAM2 probe <400> 6 cttcctgctg cagatttgga tgatt 25 <210> 7 <211> 22 <212> DNA <213> <220> <223> RdRp_F1 primer <400> 7 catacaaacc acgccaggta gt 22 <210> 8 <211> 28 <212> DNA <213> <220> <223> RdRp_R1 primer <400> 8 cttaatgtaa ggctttgtta agtcagtg 28 <210> 9 <211> 28 <212> DNA <213> <220> <223> RdRp_FAM1 probe <400> 9 attaaccttg accagggctt taactgca 28 <210> 10 <211> 25 <212> DNA <213> <220> <223> RdRp_F2 primer <400> 10 caaacataca acgtgttgta gcttg 25 <210> 11 <211> 22 <212> DNA <213> <220> <223> RdRp_R2 primer <400> 11 agttgtggca tctcctgatg ag 22 <210> 12 <211> 30 <212> DNA <213> <220> <223> RdRp_FAM2 probe <400> 12 taatgagtgt gctcaagtat tgagtgaaat 30 <210> 13 <211> 19 <212> DNA <213> <220> <223> RdRp_F4 primer <400> 13 ccttgaccag ggctttaac 19 <210> 14 <211> 22 <212> DNA <213> <220> <223> RdRp_R4 primer <400> 14 ttttaacctc tcttccgtga ag 22 <210> 15 <211> 25 <212> DNA <213> <220> <223> RdRp_FAM4 probe <400> 15 tgtaaggctt tgttaagtca gtgtc 25 <210> 16 <211> 28 <212> DNA <213> <220> <223> E_F1 primer <400> 16 ggtacgttaa tagttaatag cgtacttc 28 <210> 17 <211> 22 <212> DNA <213> <220> <223> E_R1 primer <400> 17 aatattgcag cagtacgcac ac 22 <210> 18 <211> 25 <212> DNA <213> <220> <223> E_CFO560 probe <400> 18 actagccatc cttactgcgc ttcga 25 <210> 19 <211> 23 <212> DNA <213> <220> <223> RPP30 F2 primer <400> 19 gatgcaaatc tgcaaaggaa aga 23 <210> 20 <211> 20 <212> DNA <213> <220> <223> RPP30 R2 primer <400> 20 cccaaacagc aagcctagat 20 <210> 21 <211> 24 <212> DNA <213> <220> <223> RPP30 QS670_2 probe <400> 21 taagagggcc atatgacgtg gcaa 24

Claims (30)

targeting the S gene of SARS-CoV-2,
a primer set consisting of the nucleotide sequences of SEQ ID NOs: 1 and 2; and
a probe consisting of the nucleotide sequence of SEQ ID NO: 3;
A kit for detecting SARS-CoV-2. delete delete delete delete delete delete The method according to claim 1, wherein each of the probes is labeled with one type of fluorophore selected from the group consisting of FAM, CAL Fluor Orange 560, VIC, HEX, JOE, Quasar 670 and CY5 at the 5'-end, A kit for detecting SARS-CoV-2, wherein one quencher selected from the group consisting of NFQ-MGB, BHQ-1 BHQ1 nova and BHQ-2 is labeled at the 3'-end. delete According to claim 1, wherein the primer set and probe that specifically binds to the N gene of SARS-CoV-2;
a primer set and probe that specifically binds to the RdRp gene of SARS-CoV-2; or
a primer set and probe that specifically bind to the N gene of SARS-CoV-2 and a primer set and probe that specifically bind to the RdRp gene of SARS-CoV-2;
A kit for detecting SARS-CoV-2 further comprising a. The SARS-CoV according to claim 10, wherein the primer set and probe that specifically bind to the N gene specifically bind to the region of positions 29422 to 29514 of the genomic sequence of SARS-CoV-2 (NCBI Accession No. NC_045512). -2 detection kit. 11. The method of claim 10, wherein the primer set specifically binding to the N gene comprises the nucleotide sequences of SEQ ID NOs: 4 and 5, and the probe specifically binding to the N gene comprises the nucleotide sequence of SEQ ID NO: 6, SARS -CoV-2 detection kit. delete 11. The method of claim 10, wherein the primer set and probe specifically binding to the RdRp gene is a region of positions 14106 to 14241, positions 13963 to 14119, or position 14177 of the genomic sequence of SARS-CoV-2 (NCBI accession number NC_045512). to 14283, a kit for detecting SARS-CoV-2. 11. The method of claim 10, wherein the primer set specifically binding to the RdRp gene
a primer set comprising the nucleotide sequences of SEQ ID NOs: 10 and 11; or
and a primer set comprising the nucleotide sequences of SEQ ID NOs: 13 and 14;
A probe that specifically binds to the RdRp gene comprises the nucleotide sequence of SEQ ID NO: 12 or 15, a kit for detecting SARS-CoV-2. delete The kit for detecting SARS-CoV-2 according to claim 1, further comprising a primer set and a probe binding to the E gene, which is a common region of SARS-CoV-1 and SARS-CoV-2. 18. The method of claim 17, wherein the primer set binding to the E gene comprises the nucleotide sequences of SEQ ID NOs: 16 and 17, and the probe binding to the E gene comprises the nucleotide sequence of SEQ ID NO: 18. kit. The kit for detecting SARS-CoV-2 according to claim 1, further comprising a primer set and a probe that bind to the human RPP30 (RNase P) gene as an internal control. The SARS-CoV-2 according to claim 19, wherein the primer set binding to the human RPP30 gene comprises the nucleotide sequences of SEQ ID NOs: 19 and 20, and the probe binding to the human RPP30 gene comprises the nucleotide sequence of SEQ ID NO: 21. detection kit. According to claim 1, further comprising Taq DNA polymerase, SARS-CoV-2 detection kit. The kit for detecting SARS-CoV-2 according to claim 1, wherein the kit uses a dUTP and uracil-DNA N-glycosylase (UNG) system for preventing cross-contamination. A SARS-CoV-2 detection method comprising the steps of:
(a) extracting RNA from the isolated biological sample;
(b) treating the extracted RNA with the kit of claim 1 to perform reverse transcription and polymerase chain reaction (PCR) to amplify; and
(c) confirming the result of the PCR amplification with fluorescence. The method of claim 23, wherein the isolated biological sample is at least one selected from the group consisting of nasopharyngeal or oropharyngeal swab, nasopharyngeal aspirate, bronchoalveolar lavage fluid and sputum, SARS-CoV-2 detection method. The method of claim 23, wherein the PCR is real-time PCR. The method according to claim 23, further comprising the step of (d) confirming the PCR amplification result by measuring a cycle threshold (Ct) value. A SARS-CoV-2 detection method comprising the steps of:
(a) extracting RNA from the isolated biological sample;
(b) treating the extracted RNA with the kit of claim 10 to perform reverse transcription and PCR (polymerase chain reaction) to amplify; and
(c) confirming the result of the PCR amplification with fluorescence. The method of claim 27, wherein the isolated biological sample is at least one selected from the group consisting of nasopharyngeal or oropharyngeal swab, nasopharyngeal aspirate, bronchoalveolar lavage fluid and sputum, SARS-CoV-2 detection method. The method of claim 27, wherein the PCR is real-time PCR. The method according to claim 27, further comprising the step of (d) confirming the PCR amplification result by measuring a cycle threshold (Ct) value.

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