CN115461476A

CN115461476A - Primer for detecting SARS-CoV-2 novel coronavirus and its kit, detection method and application

Info

Publication number: CN115461476A
Application number: CN202180024711.0A
Authority: CN
Inventors: 崔占峰; 黄巍; 王允; 王珲
Original assignee: Oxford University Suzhou Technology Co ltd
Current assignee: Oxford University Suzhou Technology Co ltd
Priority date: 2020-03-27
Filing date: 2021-03-26
Publication date: 2022-12-09
Also published as: WO2021190633A1; CN113444831A

Abstract

The present disclosure provides primers and kits for SARS-CoV-2 gene amplification, which, in combination with a gene amplification system including LAMP or RT-LAMP, etc., are capable of determining COVID-19 patients, asymptomatic carriers, and determining the presence or absence of SARS-CoV-2 virus, etc., in environmental samples, without the need for complex instruments, and optionally can read the test results with the naked eye, thus being suitable for large-scale SARS-CoV-2 virus screening. The disclosure also provides a use method and application of the primer and the kit.

Description

Primer for detecting SARS-CoV-2 novel coronavirus and its kit, detection method and application

Cross-referencing

The present disclosure claims priority of chinese invention patent application entitled "primers for detecting SARS-CoV-2 novel coronavirus and kits, methods and uses thereof" filed 3, 27/2020, under application number CN202010232072.4, which is hereby incorporated by reference in its entirety into the present disclosure.

Technical Field

The present disclosure relates to primers for nucleic acid detection and applications thereof, and in particular, to primers, primer sets, kits, detection methods, and applications for SARS-CoV-2 detection.

Background

The new coronavirus SARS-CoV-2 (also known as 2019-nCoV, HCoV-19 or new coronavirus), which is currently prevalent worldwide, has caused a large population to be infected with the new coronavirus pneumonia COVID-19 (also known as "new coronavirus"). Based on epidemiological investigation, the latent period of COVID-1 new coronary pneumonia is generally 1-14 days, mostly 3-7 days, symptoms mainly comprise fever, dry cough and hypodynamia, severe patients mostly have dyspnea and/or hypoxemia after one week of onset, and severe patients can rapidly progress to acute respiratory distress syndrome, septic shock, metabolic acidosis which is difficult to correct, coagulation dysfunction, multiple organ failure and the like. In order to contain the spread of viruses, the WHO urgently requires the expansion of potential patient screening and detection.

Viral nucleic acid detection, i.e., detection of viral RNA, is an effective method for viral detection. The most common nucleic acid diagnostic method at present is based on real-time RT-PCR to detect new coronaviral nucleic acid positives. For example, the Chinese Woods gene (BGI, https:// www.bgi.com/kit) and the United states center for disease control (CDC, https:// www.cdc.gov/coronavirus/2019-ncov/about/testing. Html) all provide reagents, primers and probes to support RT-PCR diagnosis of SARS-CoV-2. Currently, methods for detecting SARS-CoV-2 are either based on the detection of viral RNA itself using reverse transcription real-time polymerase chain reaction (RT-qPCR) techniques or on the detection of specific immunoglobulins IgM and IgG that appear in the blood of patients several days after infection, and these existing methods require laboratories equipped with specialized detection instrumentation, are handled by skilled scientists and technicians, and take a long time (about 2 hours or more), thus limiting the widespread use of nucleic acid detection in the current enormous detection requirements for global COVID-19 pandemics. In addition, the existing nucleic acid detection reagent has the problem of high false negative rate (the positive detection rate can be as low as 30-50%).

Disclosure of Invention

The present disclosure provides a rapid, simple, highly specific SARS-CoV-2 virus detection method.

In a first aspect of the present disclosure, there is provided an oligonucleotide primer set (hereinafter referred to as N15 primer set) for amplifying a SARS-CoV-2 gene, particularly preferably a nucleocapsid protein gene region thereof, comprising: a forward primer: SEQ ID No.1; reverse primer: SEQ ID No.2; forward inner primer: SEQ ID No.3; reverse inner primer: SEQ ID No.4; forward loop primer: SEQ ID No.5; and a reverse loop primer: SEQ ID No.6.

In a second aspect of the present disclosure, there is provided an oligonucleotide primer set (hereinafter abbreviated as O117 primer set) for amplifying a SARS-CoV-2 gene, particularly preferably an Orf1ab gene region thereof, comprising: a forward primer: SEQ ID No.7; reverse primer: SEQ ID No.8; forward inner primer: SEQ ID No.9; reverse inner primer: SEQ ID No.10; forward loop primer: SEQ ID No.11; and a reverse loop primer: SEQ ID No.12.

In a third aspect of the present disclosure, there is provided an oligonucleotide primer set (hereinafter referred to as N1 primer set) for amplifying a SARS-CoV-2 gene, particularly preferably a nucleocapsid protein gene region thereof, comprising: a forward primer: SEQ ID No.13; reverse primer: SEQ ID No.14; forward inner primer: SEQ ID No.15; reverse inner primer: SEQ ID No.16; forward loop primer: SEQ ID No.17; and a reverse loop primer: SEQ ID No.18.

In a fourth aspect of the present disclosure, there is provided an oligonucleotide primer set (hereinafter, abbreviated as S17 primer set) for amplifying a SARS-CoV-2 gene, particularly preferably a spike protein gene region thereof, comprising: a forward primer: SEQ ID No.19; reverse primer: SEQ ID No.20; forward inner primer: SEQ ID No.21; reverse inner primer: SEQ ID No.22; forward loop primer: SEQ ID No.23; and a reverse loop primer: SEQ ID No.24.

In a fifth aspect of the present disclosure, there is also provided a primer set for amplifying a SARS-CoV-2 gene, comprising a combination of any two or more of the above oligonucleotide primer sets.

In the sixth aspect of the present disclosure, a kit for detecting SARS-CoV-2, the kit comprising any one of the above-described oligonucleotide primer sets or primer set sets.

In a seventh aspect of the present disclosure, there is also provided a method for detecting SARS-CoV-2 using any one of the above-mentioned oligonucleotide primer sets or primer set sets, the method comprising: obtaining a sample to be detected; extracting RNA of a sample to be detected as a template; performing reverse transcription on the RNA and amplifying DNA obtained by the reverse transcription by using any one of the oligonucleotide primer sets or the primer set, or performing reverse transcription amplification on the RNA by using any one of the oligonucleotide primer sets or the primer set; and determining whether the sample to be tested contains SARS-CoV-2 based on the amplification result or reverse transcription amplification result.

In an eighth aspect of the present disclosure, there is also provided a use of any one of the above-mentioned oligonucleotide primer sets or primer set sets in the preparation of a reagent for detecting SARS-CoV-2.

In a ninth aspect of the present disclosure, the present disclosure also provides a method for detecting a nucleic acid in one step, the method comprising: obtaining a sample to be detected, and collecting cells from the sample to be detected; performing on said cells in one step in the same reaction system using primers suitable for detecting said nucleic acid: cell lysis; extracting nucleic acid; gene amplification or reverse transcription amplification; and determining whether the sample to be tested contains the nucleic acid based on the amplification result.

In addition, the disclosure also provides the use of the aforementioned oligonucleotide primer set, or kit, or detection method for diagnosing a SARS-CoV-2 related disease or symptom.

Drawings

FIG. 1 shows the RNA profile of SARS-CoV-2 virus and the relevant gene regions designed according to the N15, O117, N1, S17 primer set described in the first to fourth aspects of the disclosure;

FIG. 2 shows the gel electrophoresis and fluorescence results of in vitro LAMP reaction of SARS-CoV-2 synthetic DNA fragments with primer set N15, O117, N1, S17 in one embodiment according to the disclosure, wherein:

FIG. 2A shows the results of gel electrophoresis and fluorescence of N1 primer set (targeting N gene in vitro transcription DNA): (1) N1 primer set + N gene DNA (200 k copies); (2) N1 primer set + N gene DNA (200 copies); (3) N1 primer set + N gene DNA (200 k copies) + human genome; (4) N1 primer group + N gene DNA (200 copies) + human genome; (5) N1 primer group + human genome; (6) human β -actin primer + human genome; (7) Human β -actin primer + N gene DNA (200 k copies); and (L) the prestained protein Ladder;

FIG. 2B shows the results of gel electrophoresis and fluorescence of N15 primer set (targeting N gene in vitro transcription DNA): (1) N15 primer set + N gene DNA (200 k copies); (2) N15 primer set + N gene DNA (200 copies); (3) N15 primer set + N gene DNA (200 k copies) + human genome; (4) N15 primer set + N gene DNA (200 copies) + human genome; (5) N15 primer set + human genome; (6) human β -actin primer + human genome; (7) Human β -actin primer + N gene DNA (200 k copies); and (L) a prestained protein Ladder;

FIG. 2C shows the results of gel electrophoresis of the O117 primer set (targeting Orf1ab gene in vitro transcribed DNA): (1) O117 primer set + Orf1ab gene DNA (200 k copies); (2) O117 primer set + Orf1ab gene DNA (200 copies); (3) O117 primer set + Orf1ab gene DNA (20 copies); (4) O117 primer set + Orf1ab gene DNA (2 copies); (5) O117 primer set + Orf1ab gene DNA (200 copies) + human genome; (6) O117 primer set + human genome; (7) O117 primer set + water (8) human β -actin primer + human genomic DNA; and (L) a prestained protein Ladder;

FIG. 2D shows the results of gel electrophoresis of the S17 primer set (targeting S gene in vitro transcribed DNA): (1) S17 primer set + S gene DNA (200 k copies); (2) S17 primer set + S gene DNA (200 copies); (3) S17 primer set + S gene DNA (20 copies); (4) S17 primer set + S gene DNA (2 copies); (5) S17 primer set + S gene DNA (200 copies) + human genome; (6) S17 primer set + human genome; (7) S17 primer set + water (8) human beta-actin primer + human genomic DNA; and (L) a prestained protein Ladder;

FIG. 3 shows the results of gel electrophoresis of reaction mixtures sampled at 15 min (FIG. 3A), 20 min (FIG. 3B) and 30 min (FIG. 3C) in an in vitro RT-LAMP reaction targeting SARS-CoV-2 virus N gene RNA with N1 and N15 primer sets, respectively, according to one embodiment of the present disclosure: (1) N1 primer set + N gene RNA (200 copies); (2) N1 primer set + N gene RNA (20 copies); (3) N1 primer group + N gene RNA (2 copies); (4) N15 primer group + N gene RNA (200 copies); (5) N15 primer set + N gene RNA (20 copies); (6) N15 primer group + N gene RNA (2 copies); (L) prestained protein Ladder;

FIG. 4 shows gel electrophoresis results of reaction mixtures sampled at 15 min (FIG. 4A, 4D), 20 min (FIG. 4B, 4E) and 30 min (FIG. 4C, 4F) for the in vitro RT-LAMP reaction in which the SARS-CoV-2 virus Orf1ab gene RNA is targeted for the O117 primer set (FIG. 4A,4B, 4C) and the SARS-CoV-2 virus S gene RNA is targeted for the S17 primer set (FIG. 4D,4E, 4F), respectively, according to one embodiment described in the present disclosure: (1) O117 or S17 primer set + RNA target (200 k copies); (2) O117 or S17 primer set + RNA target (200 copies); (3) O117 or S17 primer set + RNA target (20 copies); (4) O117 or S17 primer set + RNA target (2 copies); (5) O117 or S17 primer set + RNA target (200 copies) + human genome; (6) O117 or S17 primer set + human genome; (7) O117 or S17 primer set + water; (8) human primer + human genome;

fig. 5 shows the results of color observation and gel electrophoresis of a colorimetric RT-LAMP reaction using the N15 primer set, where fig. 5A is the color of the reaction mixture before RT-LAMP, fig. 5B is the color of the reaction mixture after RT-LAMP, fig. 5C is the gel electrophoresis of the reaction mixture, and where (1) the N15 primer set + N gene RNA (200 k copies), according to one embodiment of the present disclosure; (2) N15 primer group + N gene RNA (200 copies); (3) N15 primer set + N gene RNA (20 copies); (4) N15 primer group + N gene RNA (2 copies); (5) N15 primer set + human genome; (6) human primer + human genome; (7) human primer + fully human RNA; and (8) the colorimetric dye MasterMix only;

FIG. 6 shows the results of RT-LAMP colorimetric/fluorescent detection in vitro experiments using fluorescently labeled primer sets of the present disclosure for SARS-CoV-2, in one embodiment according to the present disclosure, wherein FIG. 6A is a colorimetric result showing an amplified product, FIG. 6B is a fluorescent image of the product under UV light, and test tubes #1, #2, and #3 contain N15 primer set, O117 primer set, and human β actin primer set (SEQ ID Nos. 29-32) with FAM fluorescent labeling, respectively.

FIG. 7 shows RT-LAMP reaction assay results of a one-step method for detecting a virus to be detected in one embodiment of the present disclosure using a human cell line and human primers according to one embodiment of the present disclosure, (A) cells dispersed in Hanks' Balanced Salt Solution (HBSS) and containing (1) 0 cell + human β -actin primers, respectively; (2) 10 cells + human β -actin primers; (3) 50 cells + human β -actin primer; (4) 100 cells + human β -actin primers; (5) hRNA + human β -actin primers; (6) water + human β -actin primers; (7) water (without primer); and (B) cells dispersed in 0.85% NaCl solution and containing (1) 0 cells + human β -actin primers, respectively; (2) 10 cells + human β -actin primer; (3) 50 cells + human β -actin primers; (4) 100 cells + human β -actin primers; (5) hRNA + human β -actin primer; and (6) water + human β -actin primers.

Detailed Description

To facilitate an understanding of the present disclosure, the present disclosure will now be described more fully with reference to the accompanying drawings. Preferred embodiments of the present disclosure are set forth in the accompanying drawings. This disclosure may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. The terminology used in the description of the disclosure herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the disclosure. As used in this disclosure, the term "and/or" includes any and all combinations of one or more of the associated listed items.

The present disclosure provides a rapid, simple, highly specific means for detecting SARS-CoV-2 virus, which can be used to rapidly and accurately determine patients with new coronary pneumonia (including even new coronary pneumonia patients who have not yet developed an immune response), carriers with no symptoms, and to determine whether SARS-CoV-2 virus is present in other situations (e.g., environmental samples from living venues).

In the first to fourth aspects of the present disclosure, a plurality of oligonucleotide primer sets (N15 primer sets) for amplifying SARS-CoV-2 gene are provided.

In a first aspect, there is provided a N15 primer set for amplifying a SARS-CoV-2 gene, particularly preferably a nucleocapsid protein gene region thereof, the primer set comprising: a forward primer: SEQ ID No.1; reverse primer: SEQ ID No.2; forward inner primer: SEQ ID No.3; reverse inner primer: SEQ ID No.4; forward loop primer: SEQ ID No.5; and a reverse loop primer: SEQ ID No.6.

A second aspect provides an O117 primer set for amplifying the SARS-CoV-2 gene, particularly preferably the Orf1ab gene region thereof, comprising: a forward primer: SEQ ID No.7; reverse primer: SEQ ID No.8; forward inner primer: SEQ ID No.9; reverse inner primer: SEQ ID No.10; forward loop primer: SEQ ID No.11; and a reverse loop primer: SEQ ID No.12.

In a third aspect, there is provided an N1 primer set for amplifying a SARS-CoV-2 gene, particularly preferably a nucleocapsid protein gene region thereof, the primer set comprising: a forward primer: SEQ ID No.13; reverse primer: SEQ ID No.14; forward inner primer: SEQ ID No.15; reverse inner primer: SEQ ID No.16; forward loop primer: SEQ ID No.17; and a reverse loop primer: SEQ ID No.18.

In a fourth aspect, there is provided a S17 primer set for amplifying the SARS-CoV-2 gene, particularly preferably the spike protein gene region thereof, comprising: a forward primer: SEQ ID No.19; reverse primer: SEQ ID No.20; forward inner primer: SEQ ID No.21; reverse inner primer: SEQ ID No.22; forward loop primer: SEQ ID No.23; and a reverse loop primer: SEQ ID No.24.

In some embodiments, the 5' end of the forward inner primer sequence of the oligonucleotide primer set of any of the above aspects may carry a fluorescent label.

In some of these embodiments, the fluorescent label may be FAM.

In some embodiments, the Amplification using any one of the above oligonucleotide primer sets or primer set sets can be any gene Amplification technique known to those skilled in the art, including, but not limited to, polymerase Chain Reaction (PCR), multiplex Polymerase Chain Reaction (mPCR), real-Time or Quantitative Polymerase Chain Reaction (Real-Time/Quantitative PCR, qPCR), nucleic Acid Sequence-dependent Amplification (NASBA), and Loop-Mediated Isothermal Amplification (LAMP), as well as Reverse-Transcription Polymerase Chain Reaction (Reverse-Transcription Polymerase Chain Reaction, RT-PCR), reverse-Transcription Multiplex Polymerase Chain Reaction (RT-mPCR), real-Time or Quantitative Polymerase Chain Reaction (RT-qPCR), reverse-Transcription Nucleic Acid Sequence-dependent Amplification (RT-mPCR), and Reverse-Transcription Loop-Mediated Isothermal Amplification (RT-LAMP), as long as the disclosed LAMP can be achieved.

In some embodiments, the manner of amplification using the oligonucleotide primer set or primer set of any of the above aspects may be loop-mediated isothermal amplification (LAMP) or reverse transcription loop-mediated isothermal amplification (RT-LAMP).

In the sixth aspect of the present disclosure, there is also provided a kit for detecting SARS-CoV-2, the kit comprising the oligonucleotide primer set or the primer set collection of any one of the above aspects.

In some embodiments, the kit may comprise a primer set of the first and second aspects of the disclosure.

In some of these embodiments, the kit may further comprise a DNA polymerase and optionally a pH indicator.

In some embodiments, the kit may further comprise a DNA polymerase and a reverse transcriptase, and optionally a pH indicator.

In some embodiments, the kit may further comprise a single enzyme that has the dual functions of RNA reverse transcriptase and DNA polymerase.

In some embodiments, the kit may further comprise a gene amplification reaction solution or a pre-mixed solution, a negative control, a quality control, and/or instructions for use.

In some embodiments, the gene amplification reaction solution may comprise water, dNTPs, DNA polymerase (such as Taq enzyme), buffer, mgCl ₂ . In some embodiments, the gene amplification reaction solution or the premix may further include a reverse transcriptase. Optionally, the gene amplification reaction solution or the pre-mixed solution may further include an enzyme stabilizer, a fluorescent dye (e.g., an electrophoretic fluorescent dye), a pH indicator, and the like.

In some of these embodiments, the negative control can be a human actin primer. In some of these embodiments, the negative control is a human β -actin primer. In some of these embodiments, the human β -actin primers may include: a forward primer: SEQ ID No.29; reverse primer: SEQ ID No.30; forward inner primer: SEQ ID No.31; reverse inner primer: SEQ ID No.32.

In a seventh aspect of the present disclosure, there is also provided a method for detecting SARS-CoV-2 using any one of the above-mentioned oligonucleotide primer sets or primer set sets, which may comprise: obtaining a sample to be detected; extracting RNA of a sample to be detected as a template; performing reverse transcription on the RNA and amplifying DNA obtained by the reverse transcription by using any one of the oligonucleotide primer groups or the primer group sets, or performing reverse transcription amplification on the RNA by using any one of the oligonucleotide primer groups or the primer group sets; and determining whether the sample to be tested contains SARS-CoV-2 based on the amplification result or the reverse transcription amplification result.

In some of these embodiments, the detecting SARS-CoV-2 can comprise: obtaining a sample to be detected; extracting RNA of a sample to be detected as a template; performing reverse transcription on the RNA and amplifying DNA obtained by the reverse transcription by using any one of the oligonucleotide primer groups or the primer group sets, or performing reverse transcription amplification on the RNA by using any one of the oligonucleotide primer groups or the primer group sets; and determining whether the sample to be tested contains SARS-CoV-2 based on the amplification result or reverse transcription amplification result.

In some of these embodiments, the nucleic acid can be DNA. In some of these embodiments, the nucleic acid can be RNA. In some of these embodiments, the amplification may be LAMP amplification. In some of these embodiments, the reverse transcription amplification may be RT-LAMP amplification.

In some of these embodiments, the nucleic acid may be a viral RNA, and the method may comprise: obtaining a sample to be detected, and collecting cells from the sample to be detected; cell lysis, RNA extraction, RNA reverse transcription and DNA amplification are realized in one step in the same reaction system; and determining whether the sample to be tested contains the virus to be tested or not based on the amplification result. In some of these embodiments, the test sample may be a clinical viral specimen, such as a pharyngeal swab or a nasal swab.

In some embodiments, the amplification in the seventh, eighth, and ninth aspects may be LAMP, and the reverse transcription amplification may be RT-LAMP.

In some embodiments, the method for determining whether the sample to be tested contains SARS-CoV-2 based on the amplification result or the reverse transcription amplification result in the seventh, eighth, and ninth aspects can be gel electrophoresis, a fluorescent dye, or a pH indicator, or other methods for determining the quality, quantity, or semi-quantity of the amplification result. Preferably, the amplification result is confirmed by a pH indicator.

The inventors of the present disclosure developed a highly specific primer set that can rapidly, simply and sensitively detect SAV-CoV-2 by performing inventive primer design for a plurality of specific gene regions of SAV-CoV-2 virus. The primer has extremely high detection accuracy and sensitivity. Meanwhile, the inventor also overcomes the technical problem that LAMP primer design is complex and difficult (Law, J.W.F., ab Mutalib, N.S., chan, K.G., and Lee, L.H. (2015) Rapid methods for the detection of food borne bacterial pathogens: primers, applications, activities and limitations, and Frontiers in Microbiology 5), and adds a loop primer for reaction acceleration, so that the high specificity primer set can be applied to the RT-reaction, and by carrying out high-speed amplification of virus RNA, SAV-CoV-2 detection time is greatly shortened, and detection limit is remarkably reduced, and therefore, efficiency and LAMP sensitivity of SAV-CoV-2 detection are remarkably improved. In addition, in some embodiments in which the primer set of the present disclosure is used for SAV-CoV-2 detection, the result can also be determined visually by color development (e.g., pH indicator), so that the detection result can be determined on site simply and quickly without the need for laboratory specialized instruments or laboratory personnel.

The inventors of the present disclosure also provide a covi-19 detection kit using the primer set of the present disclosure, which can rapidly, simply and sensitively detect SAV-CoV-2 without complicated instruments, and optionally can read the test results with the naked eye, and thus can be applied to airports, train stations, hospitals and the like, especially regional hospitals and medical centers in rural areas, to rapidly determine new coronary pneumonia patients (including even new coronary pneumonia patients who have not yet developed immune response) and asymptomatic carriers, or to determine whether SAV-CoV-2 viruses and the like exist in homes or public places, etc., to provide crucial information for timely treatment of patients, public health decisions, and inhibition of virus propagation, and to pave the way for large-scale SARS-CoV-2 virus screening in public places and hospitals (especially regional hospitals and medical centers in rural areas).

The experimental procedures, in which specific conditions are not noted in the following examples, are generally carried out under conventional conditions or conditions recommended by the manufacturers. The various chemicals used in the examples are commercially available.

Example 1 design of specific primer sequences for detection

The inventors of the present disclosure designed highly specific primer sequences for SARS-CoV-2 virus genome sequence (GenBank, NC-045512.2) published in NCBI. In particular, the present disclosure designed 4 primer sets, i.e., O117, S17, N1, and N15 primer sets, specific for different regions in the viral gene sequence. Each primer set comprises 6 primers, namely a forward primer F3, a reverse primer B3, a forward inner primer FIP, a reverse inner primer BIP, a forward loop primer LF and a reverse loop primer LB. Primer design software PrimeExplorer (http://primerexplorer.jp/e/) As an aid.

The primer design of the present disclosure takes into account the key characteristics highly related to SARS-CoV-2 viral RNA itself to ensure high specificity, accuracy and sensitivity of each primer set. SARS-CoV-2 is a single-stranded RNA virus of approximately 30 kb. The primer sets disclosed in the present disclosure are specific for two regions of the SARS-CoV-2 virus RNA encoding Open Reading Frame 1ab (Open Reading Frame 1ab, or Orf1 ab), spike Glycoprotein (or S Protein) gene and Nucleocapsid Protein (or N Protein) gene (FIG. 1). The Orf1ab of SARS-CoV-2 was approximately 21kb and encodes the replicase polyprotein, and the inventors designed an O117 primer set against Orf1ab to cover the reverse region of the 5' end of the viral RNA (FIG. 1). The S17 primer group targets the S gene, and the spike glycoprotein encoded by the gene is a key factor for SARS-CoV-2 virus to bind to human Angiotensin Converting Enzyme 2 (ACE2 protein, angiotensin I Converting Enzyme 2) and invade human cells. In addition, the N gene of the nucleocapsid protein is located at the 3' end of the viral RNA and is a conserved sequence of SARS-like coronavirus; in the sampling and RNA extraction process, the virus RNA is attacked by RNase and degraded from the 5 'end to the 3' end, and the inventor also designs N1 and N15 primer sets aiming at the region, wherein the primer sets can detect partial degraded RNA of SARS-CoV-2.

The SARS-CoV-2 virus RNA region targeted by each primer set of the present disclosure is 240-260bp, and can be detected by gene amplification reaction.

In addition, the primer design disclosed by the invention is also suitable for application of LAMP and RT-LAMP reactions. LAMP and RT-LAMP reactions are carried out at a constant temperature (usually 65 ℃), DNA amplification is completed in as short as 30 minutes, and the required equipment is very simple. Since SARS-CoV-2 is an RNA virus of about 30kb, a common single reaction of RT and LAMP can significantly shorten the reaction time and omit the RNA purification step, so that SARS-CoV-2 can be rapidly detected. In this regard, the inventors include a forward loop primer LF and a reverse loop primer LB in each primer set for accelerating the LAMP reaction, and thus each primer set of the present disclosure can be advantageously applied to a LAMP or RT-LAMP reaction system for efficient amplification, thereby achieving rapid detection while ensuring high specificity.

The sequences of the primer sets designed by the present disclosure are shown in table 1 below.

In some embodiments, the 5' end of the forward inner primer FIP of each primer set can be fluorescently labeled (e.g., FAM-labeled) to allow rapid and convenient observation of the detection result.

TABLE 1 SARS-CoV-2 Virus detection primers

* In some embodiments, the 5' end of the forward inner primer FIP is fluorescently labeled with FAM.

Example 2: simulation experiment for in vitro detection of SARS-CoV-2 virus synthetic DNA fragment target

This example is an in vitro (in vitro) assay simulation experiment for testing primers of the present disclosure for SARS-CoV-2 synthetic DNA fragments.

1. Synthesis and purification of DNA targets

For the simulation experiments, DNA fragments were designed for four relevant regions of the N, S and Orf1ab target genes, each fragment containing the T7 promoter for in vitro transcription, i.e., N1-T7, N15-T7, S17-T7, O117-T7, and the sequences (SEQ ID NO. 25-28) are shown in Table 2. Human β -actin primers (Poon LLM, et al. Detection of human influenza A viruses by loop-mediated isothermal amplification. Journal of Clinical Microbiology 43,427-430 (2005)) were used as negative controls, the sequences (SEQ ID NO. 29-32) of which are shown in Table 3. All designed primers and DNA fragments, as well as the Positive Control plasmid (2019-nCoV _ N _ Positive Control) were prepared by Integrated DNA Technologies, IDT, UK.

TABLE 2 Synthesis of DNA fragments from N, S containing the T7 promoter (bold) and Orf1ab target Gene

TABLE 3 human beta-actin plasmid primer sequences

The DNA sequences of the N, S and Orf1ab target genes were prepared by IDT synthesis and reconstituted to 50 ng/. Mu.l according to the manufacturer's instructions. The copy number of each target gene was determined based on its molecular weight, and diluted to 200,000 copies/. Mu.l, 200 copies/. Mu.l, 0 copies/. Mu.l, and 2 copies/. Mu.l for subsequent experiments.

The DNA sequences of T7_ N, T7_ S and T7_ O were prepared by IDT synthesis and reconstituted to 50 ng/. Mu.l according to the manufacturer' S instructions.

2. LAMP experiment

1. Design of Experimental treatment groups

The experimental treatment groups for the O117, S17, N1 and N15 primer sets were set up as follows, respectively, where 200k (200,000), 200, 20 and 2, etc. represent the total copy number of the DNA sequence in the reaction mixture:

primer set for N1 (targeting N gene synthetic DNA fragment): (1) N1 primer set + N gene DNA (200 k copies); (2) N1 primer set + N gene DNA (200 copies); (3) N1 primer set + N gene DNA (200 k copies) + human genome; (4) N1 primer group + N gene DNA (200 copies) + human genome; (5) N1 primer group + human genome; (6) human β -actin primer + human genome; (7) Human β -actin primer + N gene DNA (200 k copies); and (L) a prestained protein Ladder.

N15 primer set (targeting N gene synthetic DNA fragment): (1) N15 primer set + N gene DNA (200 k copies); (2) N15 primer set + N gene DNA (200 copies); (3) N15 primer set + N gene DNA (200 k copies) + human genome; (4) N15 primer set + N gene DNA (200 copies) + human genome; (5) N15 primer set + human genome; (6) human β -actin primer + human genome; (7) Human β -actin primer + N gene DNA (200 k copies); and (L) a prestained protein Ladder.

C.o117 primer set (targeting Orf1ab gene synthesis DNA fragment): (1) O117 primer set + Orf1ab gene DNA (200 k copies); (2) O117 primer set + Orf1ab Gene DNA (200 copies); (3) O117 primer set + Orf1ab Gene DNA (20 copies); (4) O117 primer set + Orf1ab gene DNA (2 copies); (5) O117 primer set + Orf1ab gene DNA (200 copies) + human genome; (6) O117 primer set + human genome; (7) O117 primer set + water; (8) human β -actin primer + human genomic DNA; and (L) a prestained protein Ladder.

S17 primer set (target is S gene synthesis DNA fragment): (1) S17 primer set + S gene DNA (200 k copies); (2) S17 primer set + S gene DNA (200 copies); (3) S17 primer set + S gene DNA (20 copies); (4) S17 primer set + S gene DNA (2 copies); (5) S17 primer set + S gene DNA (200 copies) + human genome; (6) S17 primer set + human genome; (7) S17 primer set + water; (8) human β -actin primer + human genomic DNA; and (L) a prestained protein Ladder.

2. Experimental procedure

Before the experiment, all the equipment, the laminar flow cabinet and the workbench are sprayed with RNaseZap ^TM . Filter element pipette tips were used to prevent cross-contamination.

Prior to the reaction, 10 Xprimer mixtures (FIP, 16. Mu.M; BIP, 16. Mu.M; F3, 2. Mu.M; B3, 2. Mu.M; LF, 4. Mu.M; LB, 4. Mu.M) were prepared with the disclosed O117, S17, N1 and N15 primer sets, respectively.

Using a WarmStart ^TM LAMP 2XMaster Mix(DNA&RNA) premix (New England Biolabs, UK) for LAMP reaction. Specifically, 25. Mu.l of the reaction mixture (2 XMasterMix, 12.5. Mu.l; 10 Xprimer mix, 2.5. Mu.l; DNA target, 1. Mu.l; molecular grade water without DNase and RNase, 9. Mu.l) was mixed uniformly and centrifuged for 1 second. The LAMP reaction was carried out for 30 minutes at 65 ℃ in a thermal cycler. The amplification results were confirmed by gel electrophoresis.

As an example, a fluorescent dye (New England Biolabs, UK) was also added to the N1 and N15 partial treatment groups, and the color change was directly observed by naked eyes from the PCR tube under UV irradiation, compared with the results of gel electrophoresis.

3. The experimental results are as follows:

the results of the experiment are shown in FIGS. 2A-2D.

As shown by the results of gel electrophoresis shown in FIGS. 2A and 2B, the N1 and N15 primer sets amplified the DNA of the N gene (lanes 1-2 in FIGS. 2A and 2B), but did not amplify the human genomic DNA (lane 5 in FIGS. 2A and 2B). Similarly, as shown in FIGS. 2C and 2D, the O117 and S17 primer sets also specifically amplified the Orf1ab and S gene DNA, respectively (lanes 1-4 in FIGS. 2C and 2D), but did not amplify the human genomic DNA (lane 5 in FIGS. 2C and 2D). From this, it was found that the primer sets O117, S17, N1 and N15 all had excellent amplification specificity for SARS-CoV-2 synthetic DNA fragment target over the human genome.

Furthermore, even if a human genome is added to viral DNA, the amplification performance of the corresponding gene in the reaction mixture (lane 4 in fig. 2A and 2B, and lane 5 in fig. 2C and 2D) is not affected by interference by each primer set, showing that the primer set of the present disclosure is excellent in anti-interference, which is an essential property for detecting viral RNA from human samples using LAMP.

When the PCR tubes were observed under UV irradiation for the part of the treatment groups to which the fluorescent dye was added, it was found that the fluorescence intensity in the amplification-positive tubes was stronger than that in the amplification-negative tubes, despite the existence of the fluorescent background in the amplification-negative tubes (FIGS. 2A and 2B). The fluorescence report results are consistent with those of the electrophoresis gel.

In this example, all LAMP reactions for DNA amplification were 30 minutes. It can be seen that each primer set was able to sufficiently amplify at least 200 copies of the corresponding gene sequence within 30 minutes and a significant positive result was observed. The O117 and S17 primer sets can amplify virus DNA containing as little as 20 and 2 copies, respectively, sufficiently even within 30 minutes (lane 3 in fig. 2C, lane 4 in fig. 2D) and show positive results, indicating that the primer set of the present disclosure has very high detection efficiency and detection sensitivity.

Example 3: in vitro simulation experiment of SARS-CoV-2 virus RNA target

This example is an in vitro (in vitro) simulation experiment for testing primers of the present disclosure for the detection of SARS-CoV-2 viral RNA.

1. Acquisition and purification of RNA targets

Using HiScribe ^TM T7 high-yield RNA synthesis kit (HiScribe) ^TM T7High Yield RNA Synthesis Kit, new England Biolabs, UK), the DNA sequences synthesized by IDT as described in example 1 were transcribed in vitro for T7_ N, T7_ S and T7_ O.

The transcript was purified using the RNeasy Mini Kit (Qiagen, UK) and the concentration and quality of RNA was measured using the NanoDrop nucleic acid detector.

The copy number of each target RNA was determined based on its molecular weight and diluted to 200,000 copies/. Mu.l, 200 copies/. Mu.l, 20 copies/. Mu.l, and 2 copies/. Mu.l for subsequent experiments.

Using Fast DNA ^TM The SPIN Kit (MP Biomedicals) purified the Human whole genome from a Human iPSC cell line (Human epidemic iPSC line, product No. a18945, thermo fisher Scientific Ltd) and then further purified with a DNA wash Kit (DNA clean Kit, new England Biolabs, UK).

Human whole RNA was purified from human iPSC cells using the RNeasy Mini Kit (Qiagen, UK).

2. RT-LAMP experiments

1. Design of Experimental treatment groups

The experimental treatment groups for the O117, S17, N1 and N15 primer sets were set as follows, respectively, where 200k (200,000), 200, 20 and 2, etc. represent the total copy number of the DNA sequence in the reaction mixture:

n1 and N15 primer sets (in vitro transcribed RNA targeted to the N gene): (1) N1 primer group + N gene RNA (200 copies); (2) N1 primer set + N gene RNA (20 copies); (3) N1 primer group + N gene RNA (2 copies); (4) N15 primer set + N gene RNA (200 copies); (5) N15 primer set + N gene RNA (20 copies); (6) N15 primer set + N gene RNA (2 copies); (L) Prestaining protein Ladder.

The o117 and S17 primer sets (in vitro transcribed RNA targeted to the N gene): (1) O117 or S17 primer set + RNA target (200 k copies); (2) O117 or S17 primer set + RNA target (200 copies); (3) O117 or S17 primer set + RNA target (20 copies); (4) O117 or S17 primer set + RNA target (2 copies); (5) O117 or S17 primer set + RNA target (200 copies) + human genome; (6) O117 or S17 primer set + human genome; (7) O117 or S17 primer set + water; (8) human primer + human genome.

2. Experimental procedure

The experimental procedure of this example was substantially the same as in example 2, except that the reaction mixture was sampled at 15 minutes, 20 minutes and 30 minutes, respectively, and subjected to gel electrophoresis to confirm the amplification results.

4. The experimental results are as follows:

the experimental results are shown in fig. 3 and 4.

This example evaluates the sensitivity of RT-LAMP by further diluting the copy number of RNA target per reaction from 200 to 2 (total reaction solution volume 25 μ l), and checks the reaction efficiency and detection limit by sampling the reaction mixture at 15 min, 20 min and 30 min after the start of the reaction. The experimental result shows that after RT and LAMP are integrated into one reaction, the primer groups O117, S17, N1 and N15 can still efficiently amplify the virus RNA segment with high specificity and high sensitivity.

As shown in FIG. 3, it was shown that 2 copies of target RNA could be detected within 20 minutes and 30 minutes by both the N1 and N15 primer sets (FIGS. 3A,3B, and 3C). As shown in FIG. 4, the S17 primer set detected 2S gene RNA copies in 30 minutes of RT-LAMP reaction, and the O117 primer set was able to detect 20 Ord1ab gene RNA copies in 30 minutes. The results show that the disclosed primer sets O117, S17, N1 and N15 are very sensitive and can detect viral RNA in a 30-minute RT-LAMP reaction. Among these, the use of primers N1 and S17 allowed a sensitivity of 80 copies of viral RNA per ml to be achieved.

Furthermore, it can be seen that the higher the number of RNA copies in the sample, the shorter the time required to obtain the product (FIGS. 3 and 4). When the number of copies of RNA in each reaction was 200, significant amplification results were seen in as little as 15 minutes (FIGS. 3 and 4).

Example 4: RT-LAMP colorimetric detection in vitro experiment of SARS-CoV-2

This example is an RT-LAMP colorimetric in vitro assay for testing SARS-CoV-2 viral RNA using the primer set of the present disclosure (taking N15 as an example).

1. Experimental treatment group:

the experimental treatment groups used in this example are as follows, with 200k (200,000), 200, 20 and 2, etc. representing the total copy number of the DNA sequence in the reaction mixture: (1) N15 primer group + N gene RNA (200 k copies); (2) N15 primer set + N gene RNA (200 copies); (3) N15 primer set + N gene RNA (20 copies); (4) N15 primer set + N gene RNA (2 copies); (5) N15 primer group + human genome; (6) human primer + human genome; (7) human primer + fully human RNA; and (8) the colorimetric dye MasterMix only.

2. The experimental steps are as follows:

the RNA target acquisition and purification method and RT-LAMP experimental procedure used in this example were as described in example 3, except that WarmStart was performed ^TM LAMP 2X Master Mix(DNA&RNA) premix replaced with or WarmStart ^TM Colorimetric LAMP 2X Master Mix(DNA&RNA) premix.

After the RT-LAMP reaction was carried out for 30 minutes, the color change of the reaction mixture in the PCR reaction tube was visually observed, and the amplification result was confirmed by gel electrophoresis.

3. The experimental results are as follows:

the results of the RT-LAMP colorimetric experiments of this example are shown in FIG. 5.

Since the pH of the reaction is changed by the release of pyrophosphate by nucleic acid amplification, a positive or negative result of RT-LAMP can be shown using a pH indicator. The pH indicator may be phenol red, which is pink at pH8.2-8.6 and turns yellow (positive) when the pH drops.

FIG. 5A shows the color of the reaction mixture before and after 30 min RT-LAMP reaction, and is compared with the results of gel electrophoresis (FIG. 5B). As shown in FIG. 5A, if the target sequence has been amplified (amplification positive), a visible color change from pink to yellow can be observed. The intensity of RT-LAMP product in the gel as shown in FIG. 5B is consistent with the color change of the reaction mixture in FIG. 5A. Thus, the colorimetric RT-LAMP assay can reliably detect the N gene of SARS-CoV-2 viral RNA, and a color change can semi-quantitatively indicate the number of target sequences. Therefore, RT-LAMP results can be visualized, and the results can be read by naked eyes without specific instruments or fluorescent dyes.

Although the N15 primer set is taken as an example in the present embodiment, and the RT-LAMP reaction is taken as an example, those skilled in the art will know that the same principle can also be similarly applied to other primer sets (O117, N1, S17 primer sets) and similarly applied to the LAMP reaction.

Example 5: RT-LAMP colorimetric/fluorescent in vitro assay for SARS-CoV-2 with fluorescently labeled primers

This example is an in vitro RT-LAMP colorimetric/fluorescence detection assay for testing SARS-CoV-2 after FAM fluorescence labeling (5 '-FAM-FIP) at the 5' end of forward inner primer FIP using the primer set of the present disclosure (taking N15 and O117 as examples) (Table 1).

1. Experimental treatment group:

in vitro transcribed RNA products of the N15 synthetic DNA fragment and O117 synthetic DNA fragment were detected with the primer set N15 (# 1), O117 (# 2) with 5' -FAM-FIP, respectively, with the human β actin primer set (# 3, SEQ ID Nos. 29-32) as a negative control and water as a Blank control (Blank).

2. The experimental steps are as follows:

the experimental procedure of this example was the same as in example 4.

After the RT-LAMP reaction was carried out for 30 minutes, the color change of the reaction mixture in the PCR reaction tube was visually observed. Further, after the reaction product was purified, observation was performed under ultraviolet light.

3. The experimental results are as follows:

the results of the RT-LAMP colorimetric experiments of this example are shown in FIG. 6.

As shown in FIG. 6A, the reaction product was visually observed as a corresponding colorimetric result for each specific primer set, wherein a positive result was shown to change the color from pink to yellow, and a negative result was shown to remain the same as the color of pink. As shown in fig. 6B, the fluorescence of the positive results of the purified product under uv light showed agreement with the colorimetric results.

The fluorescence labeling primer group further overcomes various uncertainties possibly encountered by RT-LAMP reaction in practical application, such as the influence of the difference of patient sample conditions on pH change or the influence of contrast color reading of various buffers and the like, and ensures the reliability of diagnosis results through colorimetric and fluorescence double display.

Although the N15 and O117 primer sets are taken as examples in the present embodiment, and the RT-LAMP reaction is taken as an example, one skilled in the art can know that the same principle can also be similarly applied to other primer sets (such as the N1 and S17 primer sets) and similarly applied to the LAMP reaction.

Example 6: clinical test of RT-LAMP detection of SARS-CoV-2

This example is a clinical experiment of RT-LAMP detection of SARS-CoV-2 using the primer set of the present disclosure (taking N15 and O117 as examples).

1. Collection of clinical specimens

The China center for disease prevention and control authorizes the Luo lake people Hospital in Shenzhen City to detect clinical samples of SARS-CoV-2 virus. Shenzhen, roche national Hospital ethics Committee has approved a study for rapid diagnosis of COVID-19 using clinical specimens.

A total of 16 clinical samples (8 positive and 8 negative) were collected from the patients, all of which were sampled by clinical throat swabs. The collected patient respiratory specimens (throat swabs) were immediately placed in a sterile test tube containing 3ml of Virus Transport Medium (VTM) (Health Gene Technologies co. Ltd., HGT, ningbo, china) and stored and transported in the VTM.

The virus inactivation step is carried out according to the clinical sample SARS-CoV-2 nucleic acid detection standard guideline, in the Shenzhen Luo lake people Hospital biosafety level 2 (BSL 2) medical laboratory in China, the swab sample is heated for 30 minutes at 56 ℃ to inactivate the virus.

2. Extraction of clinical sample RNA

RNA was extracted from the swab sample using an RNA extraction kit (ningbohel zeuginese technologies, inc., taiwan) on the Smart LabAssist-32 platform (Taiwan dot nanotechnology Inc., taiwan). To avoid interference of TE buffer with RT-LAMP reaction, RNA was eluted with RNase and DNase-free water.

3. Routine RT-qPCR detection experiment of SARS-CoV-2 clinical RNA sample

A commercially available 2019-nCoV RT-PCR kit (Shanghai ZJ Bio-Tech Co, ltd.) from Shanghai, china, shanghai) was used to determine whether 16 clinical specimens were positive for SARS-CoV-2 virus. Sequencing was performed using the ABI 7500 real-time PCR system (Thermo Fisher Scientific Inc., USA) according to the manufacturer's instructions. The reaction mixture (volume 25. Mu.l) used was: 5. Mu.l of RNA template; 19. Mu.l 2019-nCOV RT-PCR buffer; 1 μ l RT-PCR enzyme mix. The thermal cycling conditions were: 10 min reverse transcription at 45 ℃,3 min PCR initial activation at 95 ℃, and 45 cycles of 15s at 95 ℃ and 30s at 58 ℃.

4. RT-LAMP detection experiment of clinical RNA sample of SARS-CoV-2

RT-LAMP analysis was performed on 16 clinical specimens using a premixed detection kit.

Each kit consisted of three tubes #1, #2 and #3 containing O117, N15 and human β -actin primers, respectively (table 1). First, three tubes were filled with 5. Mu.l RNase-free water (Sigma-Aldrich Co), 12.5. Mu.l WarmStart Colorimetric Lamp 2X Master Mix premix (New England Biolabs, UK) and 2.5. Mu.l 10 Xprimer Mix (FIP, 16. Mu.M; BIP, 16. Mu.M; F3, 2. Mu.M; B3, 2. Mu.M; LF, 4. Mu.M; LB, 4. Mu.M). These kits were prepared in advance at Oxford Suzhou high Research institute (OSCAR), and then transported to Shenzhen Luo lake people Hospital using a popsicle.

To detect SARS-CoV-2 virus, 5. Mu.l of RNA extracted from a patient sample was added to tubes #1, #2 and #3, respectively. The detection kit was incubated at 65 ℃ for 30 minutes.

5. Results of the experiment

The results of the experiment are shown in table 4.

After RT-LAMP reaction, tubes #1 and #2 of each kit turned yellow in all 8 positive samples, indicating the presence of the target viral RNA in the samples, while tubes #1 and #2 remained pink in all 8 negative samples. Test tube No.3 of all test kits remained pink in both the positive and negative samples, indicating that human β -actin primers were unable to detect β -actin gene transcripts in patient samples.

The results of the conventional RT-PCR detection experiment and RT-LAMP detection experiment of SARS-CoV-2 clinical RNA samples are shown in Table 4. RT-LAMP analysis has a 100% match with the conventional RT-PCR results when diagnosing COVID-19 samples. Comparing the results of RT-qPCR and RT-LAMP, it can be seen that the Ct value of RT-qPCR to Orf1ab gene is 38, which indicates that the visual color change reading of RT-LAMP is highly sensitive.

TABLE 4 comparison of RT-PCR and RT-LAMP detection assay results for SARS-CoV-2 clinical RNA samples

Note: ct is the cycle threshold; ORF1ab: ORF1ab gene; n is N gene; e is E gene; IC internal standard, RNase P gene.

Example 7: RT-LAMP reaction experiment of one-step nucleic acid detection method

This example is an example of an RT-LAMP reaction using a human cell line and human primers as an example to examine the one-step nucleic acid detection method described in the present disclosure. One skilled in the art will recognize that the methods described in this example can also be used to detect other different cells in a variety of samples by changing the appropriate primers, enzymes, and reagents.

1. Experimental procedure

After dispersing appropriate amount of Human cell line (Human epidermal iPSC line, product No. A18945, thermoFisher Scientific Ltd) cells in HBSS and 0.85% NaCl solution, respectively, 1. Mu.l of each was used for the subsequent experiments according to the following treatment group settings.

The treatment groups for this experiment were:

a, HBSS: (1) 0 cell + human β -actin primer; (2) 10 cells + human β -actin primer; (3) 50 cells + human β -actin primer; (4) 100 cells + human β -actin primer; (5) hRNA + human β -actin primers; (6) water + human β -actin primers; (7) water (no primer); and

b.0.85% NaCl (1. Mu.l): (1) 0 cell + human β -actin primer; (2) 10 cells + human β -actin primer; (3) 50 cells + human β -actin primer; (4) 100 cells + human β -actin primer; (5) hRNA + human β -actin primers; (6) Water + human β -actin primer.

The human primers used in this example were the human β -actin primers used in the previous examples, and the reagents and RT-LAMP protocol were as described in example 4, but the reaction time was 40 minutes.

After the RT-LAMP reaction was carried out for 30 minutes, the color change of the reaction mixture in the PCR reaction tube was visually observed.

2. Results of the experiment

As shown in fig. 7A and 7B, after 30 minutes of RT-LAMP reaction of cells dispersed in HBSS and 0.85% nacl solution, the PCR tubes containing cells and human primers changed from pink to yellow, indicating that the sample cells were subjected to cell lysis, RNA extraction, RNA reverse transcription and DNA amplification in one step in the same reaction system, and positive results were visually observed under natural light, which was highly efficient and simple.

In addition, when the cell concentration is as low as 100, 50 or even 10 cells/μ l, a positive result can still be clearly observed, which indicates that the method has an extremely low detection limit and can detect the virus in the sample with high sensitivity.

The technical features of the embodiments described above may be arbitrarily combined, and for the sake of brevity, all possible combinations of the technical features in the embodiments described above are not described, but should be considered as being within the scope of the present specification as long as there is no contradiction between the combinations of the technical features.

The above-mentioned embodiments only express several embodiments of the present disclosure, and the description thereof is more specific and detailed, but not construed as limiting the scope of the invention. It should be noted that various changes and modifications can be made by one skilled in the art without departing from the spirit of the disclosure, and these changes and modifications are all within the scope of the disclosure. Therefore, the protection scope of the present disclosure should be subject to the appended claims.

Claims

An oligonucleotide primer set for amplifying SARS-CoV-2 gene, comprising:

a forward primer: SEQ ID No.1;

reverse primer: SEQ ID No.2;

forward inner primer: SEQ ID No.3;

reverse inner primer: SEQ ID No.4;

forward loop primer: SEQ ID No.5; and

reverse loop primer: SEQ ID No.6.
An oligonucleotide primer set for amplifying SARS-CoV-2 gene, comprising:

a forward primer: SEQ ID No.7;

reverse primer: SEQ ID No.8;

forward inner primer: SEQ ID No.9;

reverse inner primer: SEQ ID No.10;

forward loop primer: SEQ ID No.11; and

reverse loop primer: SEQ ID No.12.
An oligonucleotide primer set for amplifying SARS-CoV-2 gene, comprising:

a forward primer: SEQ ID No.13;

reverse primer: SEQ ID No.14;

forward inner primer: SEQ ID No.15;

reverse inner primer: SEQ ID No.16;

forward loop primer: SEQ ID No.17; and

reverse loop primer: SEQ ID No.18.
An oligonucleotide primer set for amplifying SARS-CoV-2 gene, comprising:

a forward primer: SEQ ID No.19;

reverse primer: SEQ ID No.20;

forward inner primer: SEQ ID No.21;

reverse inner primer: SEQ ID No.22;

forward loop primer: SEQ ID No.23; and

reverse loop primer: SEQ ID No.24.
An oligonucleotide primer set for amplifying SARS-CoV-2 gene, having the sequence of the oligonucleotide primer set according to any one of claims 1-4, and wherein the 5' end of the forward inner primer sequence carries a fluorescent label.
A primer set for amplifying a SARS-CoV-2 gene, comprising a combination of any two or more of the oligonucleotide primer sets according to any one of claims 1-5.
A kit for detecting SARS-CoV-2, comprising the oligonucleotide primer set according to any one of claims 1-5 or the primer set according to claim 6.
The kit according to claim 7, wherein the primer set included in the kit is the primer set according to claim 1 and the primer set according to claim 2, and the primer sets each independently carry or do not carry a fluorescent label at the 5' end of the primer sequence in the forward direction thereof.
The kit of claim 7, wherein the kit further comprises a DNA polymerase; and the kit further comprises at least one or neither of an RNA reverse transcriptase, a pH indicator.
The kit of claim 7, further comprising a single enzyme that has the dual functions of RNA reverse transcriptase and DNA polymerase.
The kit according to claim 7, wherein the kit further comprises any one or more of a gene amplification reaction solution, a negative control, a quality control material and an instruction for use.
The kit of claim 11, wherein the negative control is a human β -actin primer.
A method of detecting SARS-CoV-2 using the oligonucleotide primer set of any one of claims 1-5 or the primer set of claim 6, the method comprising:

obtaining a sample to be detected;

extracting RNA of a sample to be detected as a template;

reverse transcribing the RNA and amplifying the DNA resulting from the reverse transcription using the oligonucleotide primer set according to any one of claims 1 to 5 or the primer set according to claim 6, or reverse transcribing and amplifying the RNA using the oligonucleotide primer set according to any one of claims 1 to 5 or the primer set according to claim 6; and

and determining whether the sample to be detected contains SARS-CoV-2 or not based on the amplification result or the reverse transcription amplification result.
Use of the set of oligonucleotide primers of any one of claims 1-5 or the set of primers of claim 6 in the preparation of a reagent for the detection of SARS-CoV-2.
Use according to claim 14,

the detection of SARS-CoV-2 comprises:

obtaining a sample to be detected;

extracting RNA of a sample to be detected as a template;

reverse transcribing the RNA and amplifying the DNA resulting from the reverse transcription using the oligonucleotide primer set according to any one of claims 1 to 5 or the primer set according to claim 6, or reverse transcribing the RNA using the oligonucleotide primer set according to any one of claims 1 to 5 or the primer set according to claim 6; and

and determining whether the sample to be tested contains SARS-CoV-2 or not based on the amplification result or the reverse transcription amplification result.
A method for the one-step detection of nucleic acids, the method comprising:

obtaining a sample to be detected, and collecting cells from the sample to be detected;

using a set of oligonucleotide primers suitable for detecting the desired nucleic acid, said cells are subjected to a single step in the same reaction system: cell lysis; extracting nucleic acid; amplification or reverse transcription amplification; and

determining whether the sample to be tested contains the nucleic acid based on the amplification result.
The method of claim 16, wherein the oligonucleotide primer set is the oligonucleotide primer set of any one of claims 1-5.
Use of the set of oligonucleotide primers according to any one of claims 1-5, or the set of primer sets according to claim 6, or the kit according to claims 7-12, or the method according to claim 13, or the method according to claim 16 or 17, for diagnosing a SARS-CoV-2 associated disease or condition.