CN112080585B - Novel coronavirus (SARS-CoV-2) rapid detection kit and method thereof - Google Patents

Abstract

The invention provides a novel household rapid detection kit for coronaviruses and a method thereof, wherein the method comprises the following steps of S1: obtaining a component to be detected in a sample by adopting a magnetic bead adsorption method, and cracking the adsorbed cells or pathogens by a cracking method to release nucleic acid in the cells or pathogens; s2: performing isothermal amplification on the nucleic acid sample for a plurality of times, wherein the last isothermal amplification is isothermal amplification for generating single strands of nucleic acid or other methods for generating single strands; s3: hybridizing the single-stranded product obtained by the step S2 for multiple times at constant temperature amplification or other methods with a modified probe; s4: adding specific exonuclease into the hybridized product in the step S3, and cutting off redundant probes; s5: the product of S4 was detected. The kit comprises magnetic beads, cell lysate, isothermal amplification primers, isothermal amplification enzymes, probes, specific exonucleases and detection test paper; the kit and the method provided by the invention can be used for rapidly and accurately detecting the novel coronavirus at home.

Description

Novel coronavirus (SARS-CoV-2) rapid detection kit and method thereof

Technical Field

The invention relates to the technical field of biology, in particular to a novel rapid coronavirus detection kit and a method thereof.

Background

SARS-CoV-2 is a class of plus-sense single stranded RNA viruses. The current detection strategies are mainly divided into two types: 1. based on the detection of IgG and IgM antibodies, the detection method is suitable for a period of infection, has low applicability to the early stage of infection or the patients without symptoms infection, and has obvious limitations; 2. based on nucleic acid detection, this is currently the "gold standard". In terms of nucleic acid detection, three main categories are: (1) The conventional real-time quantitative fluorescence PCR detection is the most common method at present, one or more pairs of primers are designed aiming at a new coronavirus sequence, nucleic acid is extracted, reverse transcription is carried out, qPCR is carried out, and the detection result is the cycle number; (2) On-site detection methods that do not rely on large instruments, such as the us researchers developed CRISPR/Cas 12-based methods detect, which employ samples of nucleic acids extracted from nasal or pharyngeal swabs that do not meet the needs of patient self-sampling and self-handling; (3) Household rapid detection, i.e. a simple detection scheme in which a detector can self-sample, self-operate and self-read results.

The household detection is independent of a large instrument, and the novel coronavirus rapid detection method capable of sampling by itself can greatly improve the detection efficiency and safety of SARS-CoV-2, and provides a powerful means for epidemic prevention and control and national policy formulation. However, the prior art lacks a kit and a method for rapid and accurate rapid detection of novel coronaviruses.

Disclosure of Invention

The invention overcomes the defects of the prior art and provides a kit and a method for rapidly and accurately detecting novel coronaviruses.

In a first aspect of the present invention, there is provided a novel coronavirus (SARS-CoV-2) rapid assay kit and method thereof, comprising the steps of:

s1: obtaining a sample to be detected, obtaining a component to be detected in the sample by adopting a magnetic bead adsorption method, and cracking adsorbed cells or pathogens by a cracking method to release nucleic acid in the cells or pathogens; step S1 can be replaced by any other mode capable of obtaining sample nucleic acid, such as phenol chloroform extraction, adsorption column extraction and the like;

s2: performing multiple isothermal amplifications on the nucleic acid sample obtained in the step S1, wherein the final isothermal amplifications are performed to generate nucleic acid single strands; step S2 may be replaced by any other means capable of producing a single strand of nucleic acid, such as T7 exonuclease treatment, in vitro transcription, etc.;

s3: hybridizing the single-stranded product obtained by the step S2 through multiple isothermal amplification or other methods with a modified probe, wherein the probe type comprises a DNA probe, an RNA probe, an LNA modified probe, a peptide nucleic acid probe and the like;

s4: adding specific exonuclease into the hybridized product in the step S3, and cutting off redundant probes;

s5: the product of S4 was detected.

Preferably, step S2 further includes: and (2) adding reverse transcriptase into the RNA nucleic acid sample obtained in the step (S1) to enable the RNA nucleic acid sample to be reversely transcribed into a DNA nucleic acid sample.

Preferably, the sample in step S1 includes body fluid, mouthwash, saliva, nasal swab specimen and pharyngeal swab specimen, sputum, scraping, urine, feces, body secretions, tissue samples; still more preferably, the sample types are mouthwash, saliva, nasal swab specimens, and pharyngeal swab specimens.

Preferably, the magnetic beads used in the magnetic bead adsorption method in step S1 include concanavalin a coated magnetic beads.

Preferably, the probe in step S3 is a modified oligonucleotide probe; further preferably, the types of probe modifications include: biotin modification (Biotin), digoxigenin modification (Digoxigenin), fluorescein isothiocyanate modification (FITC), texas Red modification (Texas Red-X), phosphorylation modification (Phosphorination), amino modification (Aminorinker C6/7/12), sulfhydryl modification (thio C6/thio-C6S-S), thio modification (Phosphorothioate), fluorophore modification (FAM/HEX/ROX/CY-3/CY-5/JOE/TET etc.), quencher modification (MGB/BHQ 1/BHQ2/TAMRA etc.; still further preferably, the probe modification is: digoxin (DIGOXIGENIN) modification, BIOTIN (BIOTIN) modification.

Preferably, the base sequence of the probe is SEQ ID NO.1.

SEQ ID NO.1 CAGCCATAACCTTTCCACATACCGCAGACG。

Preferably, the specific exonuclease in step S4 comprises exonuclease VII, lambda exonuclease, T5 exonuclease, T7 exonuclease, exonuclease V, exonuclease III, recJ1 exonuclease, exonuclease I; still more preferably, the exonuclease is exonuclease VII.

Preferably, the lysis method of step S1 comprises cell lysis using a lysis solution.

Preferably, the lysate comprises 10mM HEPES,0.3%SDS,0.5%IGEPAL-CA630,1.5mM MgCl2,0.1% diethyl pyrocarbonate, 10mM KCl,50mM Tris,10mM EDTA,1%RNA enzyme inhibitor, and 1% aqueous protease solution.

Preferably, the isothermal amplification described in step S2 comprises a recombinase polymerase amplification technique, a recombinase-assisted amplification technique, a loop-mediated isothermal amplification technique, a helicase-dependent isothermal amplification technique, a strand displacement isothermal amplification technique, a nucleic acid sequence-based isothermal amplification technique, a rolling circle amplification technique, a nicking endonuclease amplification technique.

Isothermal amplification technique of recombinase polymerase (Recombinase Polymerase Amplification, RPA): the method mainly relies on three enzymes, namely recombinase capable of binding single-stranded nucleic acid (oligonucleotide primer), single-stranded DNA binding protein (SSB) and strand displacement DNA polymerase, and a protein-DNA complex formed by binding the recombinase and the primer can search for homologous sequences in double-stranded DNA. Once the primer has located the homologous sequence, a strand exchange reaction occurs to form and initiate DNA synthesis, allowing exponential amplification of the target region on the template. The replaced DNA strand binds to SSB, preventing further replacement. In this system, a synthetic event is initiated by two opposing primers. The whole process can be carried out at room temperature, and generally, a detectable level of amplification product can be obtained in about 10 minutes.

Recombinase-assisted amplification technique (RAA): a method for quickly amplifying nucleic acid at room temp features that the recombinase obtained from bacteria or fungus is used to bind the primer tightly to form a polymer, and after the primer is matched with template, the template DNA is melted by the aid of single-chain DNA binding protein and a new complementary DNA strand is formed by DNA polymerase.

Loop-mediated isothermal amplification technique (Loop-mediated isothermal amplification, LAMP): the LAMP method designs 4-6 primers for the region to be detected on the target gene, and utilizes the strand displacement DNA polymerase to carry out amplification reaction under the constant temperature condition, so that the amplification can be realized within 15-60 minutes at the temperature of 60-65 ℃, and a large amount of amplification products can be generated by the reaction.

Isothermal amplification technique of strand displacement: compared with the traditional method, the strand displacement isothermal amplification technology based on the notch enzyme, which is established by the technology, is simpler, more convenient, faster, environment-friendly and low in price, has very strong practical application potential, and is particularly suitable for field detection.

Rolling circle amplification: the rolling ring type replication is to synthesize new DNA ring molecules by using a piece of ring DNA as a template. A nick is first created in the replication origin of one single strand, and then new single strands are continuously synthesized using the other single strand as a template. The released newly synthesized single strand is or is first duplicated into double-stranded DNA, and after being cut into unit length by enzyme, circular double-stranded DNA molecule is formed; or the released newly synthesized single-stranded DNA is cut into single-stranded circular DNA molecules in unit length by enzyme and then copied into double-stranded circular DNA molecules.

Isothermal amplification techniques based on nucleic acid sequences (Nucleic acid sequence-based amplification, NASBA): nucleic acid sequence dependent amplification technology (NASBA) is an enzymatic process of specific in vitro isothermal amplification of nucleotide sequences mediated by two primers using RNA in the nucleic acid sequence as template. The whole reaction consists of a non-circulating phase and a circulating phase, wherein the non-circulating phase is firstly performed, under the action of AMV reverse transcriptase, primer 1 and template RNA are annealed to synthesize cDNA, RNA/DNA hybrid is formed, then RNaseH degrades RNA, primer 2 and cDNA are annealed to synthesize a second DNA complementary strand. The formed DNA double strand recognizes the promoter sequence by T7 RNA polymerase, catalyzes and synthesizes RNA, enters the circulation phase and carries out mass amplification on the template.

Helicase-dependent isothermal amplification technique (Helicase-dependent isothermal DNA amplification, HDA): the technology simulates the natural process of in vivo DNA replication, unwinds DNA double chains at constant temperature by using helicase, uses the DNA single chain binding protein to stabilize the unwound single chains to provide templates for primers, synthesizes complementary double chains under the action of DNA polymerase, and continuously repeats the cyclic amplification process to finally realize the exponential growth of target sequences.

The nicking endonuclease amplification reaction (nicking endonuclease amplification reaction, NEAR) is a strand displacement amplification technique in which, at the site of a nick formed by nicking with a nicking endonuclease, dNTPs are polymerized and extended from the 3' -end of the nick by the action of a polymerase to displace an isostatically positioned DNA strand, thereby forming a new complete DNA sequence containing the nicking endonuclease recognition site. This double strand is again cleaved by recognition by the endonuclease, and the "polymerization-nicking" cycle is initiated, producing a large number of displaced DNA single strands, resulting in exponential amplification.

Preferably, the multiple isothermal amplifications described in step S2 include one recombinase polymerase amplification and one nicking endonuclease amplification in sequence.

Preferably, the nicking endonuclease used for the nicking endonuclease amplification is Nb.BtsI, nt.BspQI, nt.CviPII, nt.BstNBI, nb.BsrDI, nb.BtsI, nt.AlwI, nb.BbvCI, nt.BbvCI, nb.BsmI, nb.BssSI, nt.Bssmai, and the strand displacing nucleic acid polymerase used for the nicking endonuclease amplification is Bst3.0 DNA polymerase, bst2.0 DNA polymerase, klenow DNA polymerase, phi29 DNA polymerase; still more preferably, the nicking enzyme used for nicking enzyme amplification is nb.btsi and the strand displacing nucleic acid polymerase used for nicking enzyme amplification is bst3.0 DNA polymerase.

Preferably, the forward primer has an alkaline sequence comprising the nicking site CACTGC and protecting base of the nicking endonuclease, and the forward primer has the sequence: SEQ ID NO.2; the reverse primer sequence is: SEQ ID NO.3.

SEQ ID NO.2 Wherein the boxes are protected bases, the single thick underline is a nicking site, and the double thin underline is a template binding sequence.

SEQ ID NO.3 CCCGTTTAAAAACGATTGTGCATCAGCTGAC。

Preferably, the method for detecting the hybridization product in step S5 includes a colloidal gold method, a colloidal carbon method; further preferably, the hybridization product is detected by a colloidal gold test strip.

In a second aspect of the invention, a novel rapid coronavirus detection kit is provided, which comprises magnetic beads, lysate, isothermal amplification primers, isothermal amplification enzymes, probes, specific exonucleases and detection test paper.

Preferably, the magnetic beads comprise concanavalin a coated magnetic beads.

Preferably, the probe contains biotin and digoxin modifications; further preferably, the base sequence of the probe is SEQ ID NO.1

Preferably, the specific exonucleases include exonuclease VII, lambda exonuclease, T5 exonuclease, T7 exonuclease, exonuclease V, exonuclease III, recJ1 exonuclease, exonuclease I; still more preferably, the exonuclease is exonuclease VII.

Preferably, the lysate comprises 10mM HEPES,0.3%SDS,0.5%IGEPAL-CA630,1.5mM MgCl2,0.1% diethyl pyrocarbonate, 10mM KCl,50mM Tris,10mM EDTA,1%RNA enzyme inhibitor, and 1% aqueous protease solution.

Preferably, the isothermal amplification enzymes comprise recombinase polymerase and nicking endonuclease; further preferably, the basic sequence of the isothermal amplification forward primer comprises a nicking site CACTGC and a protecting base of a nicking endonuclease, and the forward primer sequence is: SEQ ID NO.2; the reverse primer sequence is: SEQ ID NO.3.

Preferably, the test paper comprises a colloidal gold test strip.

In a third aspect, the invention provides the use of a kit according to the invention for the preparation of a reagent for detecting a novel coronavirus and/or for detecting a disease caused by a novel coronavirus.

Preferably, the disease caused by the novel coronavirus comprises novel coronavirus pneumonia.

Compared with the prior art, the invention has the following advantages: the invention develops a set of SARS-CoV-2 detection scheme capable of home self-detection. The early stage tests are carried out in DNA and RNA of SARS-CoV-2 standard substance, and has very high sensitivity and specificity; the detection of nucleic acid extracted from nasal and pharyngeal swabs of new patients, 5 cases with 4 cases, and 37 cycles, shows that this method has extremely high sensitivity. In addition, we develop a unique nucleic acid extraction method based on magnetic bead adsorption, and realize a complete set of processes of nucleic acid extraction, nucleic acid detection and result reading. The scheme does not depend on a large instrument, only needs two temperatures of 37 ℃ and 95 ℃ and has the condition of home detection.

Drawings

FIG. 1 shows the results of nucleic acid validation of cells in mouthwash based on magnetic bead enrichment and lysis of lysates in example 1 of the present invention;

FIG. 2 shows the result of cleavage of the probe by the probe and the single strand exonuclease in example 2 of the present invention;

FIG. 3 shows the results of the detection of novel coronavirus DNA and RNA standards in example 2 of the present invention;

FIG. 4 shows the results of the detection of purified nucleic acids in a nasal swab or pharyngeal swab of a patient with a new crown in example 3 of the present invention.

FIG. 5 shows the results of a test using a new crown patient mouthwash in example 4 of the present invention.

Detailed Description

Example 1 nucleic acid in cells was released with magnetic beads and lysate and subjected to PCR validation

1.1, rinsing with clear water;

1.2, adding 5ml of RPM 1640 culture medium, rinsing for 10s, and spitting the culture medium into a 50ml collecting tube;

1.3, sucking 1ml of mouthwash by a suction pipe, adding the mouthwash into 2ml of a B pipe containing magnetic beads, reversing and uniformly mixing, and standing for 10min at room temperature;

1.4, placing the pipe B on a magnetic rack, adsorbing for 10min, and discarding liquid;

1.5 removing the B tube from the magnetic rack, adding 100 μl of lysate, standing at room temperature for 5min at 95deg.C for 1min, taking out, cooling to room temperature, and adsorbing on the magnetic rack.

1.6, PCR verification of human cell nucleic acid: wherein the PCR template of the negative control is RPMI1640 culture medium, the PCR template of the positive control 1 is HEK293 genome DNA, and the PCR template of the positive control group 2 is cell nucleic acid obtained by adopting a commercial kit to lyse oral epithelial cells; the PCR templates of the experimental group are magnetic beads and lysate to release cellular nucleic acid.

PCR system:

PCR conditions:

wherein, the base sequence of the beta-actin-F primer is as follows: SEQ ID NO.4; the base sequence of the beta-actin-R primer is SEQ ID NO.5.

SEQ ID NO.4GGACTTCGAGCAAGAGATGG

SEQ ID NO.5：AGCACTGTGTTGGCGTACAG

1.7, 10. Mu.l of the amplified product was electrophoresed in a 2% agarose gel (containing a nucleic acid dye), and the gel was photographed.

Results: the electrophoresis results of the experimental group, the negative control group, the positive control group 1 and the positive control group 2 are shown in the attached figure 1. The above results prove that the magnetic beads and the lysate method provided by the invention can release nucleic acid in cells, and the effect of releasing nucleic acid is almost the same as that of a commercial kit.

Example 2 detection of novel coronavirus (SARS-CoV-2) Probe degradation assay

2.1, preparing a probe, designing and modifying the probe, wherein the nucleic acid sequence of the probe is as follows: SEQ ID NO.1, wherein the 5 'end is marked with digoxin modification and the 3' end is marked with biotin modification.

2.2, negative control group (sample 1) was water, experimental groups were: 1pmol of the modified probe (sample 2), 10pmol of the modified probe (sample 3), and 10pmol of the modified probe were digested with single strand exonuclease Exo VII (sample 4).

2.3, 50 mu l of the product in 2.2 is taken, a colloidal gold test strip is inserted into the product for standing chromatography, and the result is read within 5 minutes.

2.4, the results are shown in the figure 2, and the positions of the quality control lines (C) are all provided with strip displays, so that the experiment is successful; at the test line (T) position, sample 1 has no band negative, sample 2 has a band positive, sample 3 has a band positive, and sample 4 has no band negative. The detection of the probe can be directly realized, and the probe can not be detected after being cut by the exonuclease.

EXAMPLE 3 detection of novel coronavirus (SARS-CoV-2) DNA Standard and RNA Standard

3.1, preparing a detection sample: wherein the negative control group (sample 1) adopts water, and the experimental group adopts 1000 copies of SARS-CoV-2DNA fragment (sample 2), 10000 copies of SARS-CoV-2RNA standard (sample 3) and 1000 copies of SARS-CoV-2RNA standard (sample 4) respectively.

3.2, first isothermal amplification: and (3) using the detection sample obtained in the step (1) as a template, and using an RNA isothermal rapid amplification KIT (WLRN 8206KIT in the Anpul future) to carry out isothermal amplification, wherein the isothermal amplification step refers to the examination KIT instruction book.

The reaction system is as follows:

reaction conditions: 37℃for 12min.

Wherein, the forward reactant F is: SEQ ID NO.2;

the reverse primer R is: SEQ ID NO.3.

3.3, secondary amplification: 1. Mu.l of Nb.BtsI, 1. Mu.l of Bst3.0, 4. Mu.l of dNTPs were added and reacted at 37℃for 15min and at 95℃for 5min.

3.4, probe hybridization: mu.l of 10. Mu.M labeled probe (detection probe provided in example 2) was added and hybridization was performed at 95℃for 1min, followed by slow cooling to room temperature (about 15 min).

3.5, degradation of redundant probes: mu.l of the post-hybridization product was taken and 0.5. Mu.l of Exo VII was added thereto at 37℃for 5min.

3.6, detecting a colloidal gold test strip: 60 μl ddH2O was added, and after mixing, a colloidal gold test strip was inserted into the liquid, and the result was read within 5 minutes.

3.7, the results are shown in the figure 3, and the positions of the quality control lines (C) are all provided with strip displays, so that the experiment is successful; at the test line (T) position, no band of sample 1 is negative, no band of sample 2 is positive, no band of sample 3 is positive, and no band of sample 4 is positive. The method provided by the invention can detect SARS-CoV-2DNA and SARS-CoV-2RNA, and the detection accuracy is 100%.

Example 4 detection of novel coronavirus (SARS-CoV-2) by clinical pharyngeal swab sample

4.1, collecting throat swab samples of 5 cases of novel coronavirus pneumonia suspected patients, wherein the clinical nucleic acid diagnosis results are as follows: sample 1, positive; sample 2, negative; sample 3, negative; sample 4, positive; sample 5, negative.

4.2 nucleic acid was extracted from a throat swab sample using the method provided in example 1.

4.3, using water as negative control, 1000 copies of SARS-CoV-2DNA fragment as positive control, 5 cases of patient nucleic acid obtained in step 4.1 as experimental groups, and performing detection by the method of example 3.

4.4, the results are shown in the figure 4, and the positions of the quality control lines (C) are all provided with strip displays, so that the experiment is successful; at the position of the detection line (T), the negative control has no strip and is negative, the positive control has a strip and is positive, the sample 1 has a strip and is positive, the sample 2 has no strip and is negative, the sample 3 has no strip and is negative, the sample 4 has a strip and is positive, and the sample 5 has no strip and is negative. The result of the method provided by the invention for detecting suspected patients is completely consistent with the clinical detection result, which shows that the method has the potential of popularization and application for detecting novel coronaviruses.

Example 5 detection of novel coronavirus (SARS-CoV-2) by mouthwash sample

5.1, collecting mouthwash samples of 4 cases of novel patients diagnosed with coronavirus pneumonia, wherein the clinical nucleic acid diagnosis results are as follows: sample 1, positive; sample 2, positive; sample 3, positive; sample 4, positive.

5.2 nucleic acid in a mouthwash sample was extracted using the method provided in example 1.

5.3, using water as negative control, 1000 copies of SARS-CoV-2DNA fragment as positive control, and 4 cases of patient nucleic acid obtained in step 5.1 as experimental groups, the method of example 3 was used for detection.

5.4, the results are shown in the figure 5, and the positions of the quality control lines (C) are all provided with strip displays, so that the experiment is successful; at the position of the detection line (T), the negative control has no band and is negative, the positive control has a band and is positive, the sample 1 has a band and is positive, the sample 2 has a band and is negative, the sample 3 has a band and is positive, and the sample 4 has a band and is positive. The result of the method for detecting suspected patients is completely identical with the clinical detection result, which proves that the method has the potential of popularization and application of detecting novel coronaviruses.

The above description of the specific embodiments of the present invention has been given by way of example only, and the present invention is not limited to the above described specific embodiments. Any equivalent modifications and substitutions for the present invention will occur to those skilled in the art, and are also within the scope of the present invention. Accordingly, equivalent changes and modifications are intended to be included within the scope of the present invention without departing from the spirit and scope thereof.

Sequence listing

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SHANGHAI EPIPROBE BIOTECHNOLOGY Co.,Ltd.

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Claims (21)

1. A novel rapid detection method for coronaviruses with non-diagnostic purposes, which is characterized by comprising the following steps:

s1: obtaining a sample to be detected, obtaining a component to be detected in the sample by adopting a magnetic bead adsorption method, and cracking the adsorbed cells by a cracking method to release nucleic acid in the cells;

s2: performing multiple isothermal amplifications on the nucleic acid sample obtained in the step S1, wherein the final isothermal amplifications are performed to generate nucleic acid single strands;

s3: hybridizing the single-stranded product amplified in the step S2 at multiple constant temperature with the modified probe;

s4: adding specific exonuclease into the hybridized product in the step S3, and cutting off redundant probes;

s5: detecting the product of S4;

wherein, the magnetic beads used in the magnetic bead adsorption method in the step S1 comprise concanavalin A coated magnetic beads;

the method for detecting the hybridization product in step S5 includes a colloidal gold method and a colloidal carbon method.

2. The method for rapid detection of a novel coronavirus of non-diagnostic interest as recited in claim 1, wherein step S2 further comprises: and (2) adding reverse transcriptase into the RNA nucleic acid sample obtained in the step (S1) to enable the RNA nucleic acid sample to be reversely transcribed into a DNA nucleic acid sample.

3. The method according to claim 1, wherein the probe in step S3 is a modified oligonucleotide probe modified with one or more of biotin, digoxin, texas red, phosphorylating, amino, thiol, thio, fluorescent, or quencher.

4. The method for rapid detection of coronavirus of non-diagnostic interest as claimed in claim 3, wherein the probe has a base sequence of SEQ ID NO.1.

5. The method according to claim 1, wherein the specific exonuclease in step S4 is one or more of exonuclease VII, lambda exonuclease, T5 exonuclease, T7 exonuclease, exonuclease V, exonuclease III, recJ1 exonuclease, and exonuclease I.

6. The method according to claim 1, wherein the lysis method of step S1 comprises cell lysis using a lysate.

7. The method for rapid detection of novel coronaviruses of non-diagnostic interest as recited in claim 6, wherein said lysate comprises 10mM HEPES,0.3%SDS,0.5%IGEPAL-CA630,1.5mM MgCl ₂ 0.1% diethyl pyrocarbonate, 10mM KCl,50mM Tris,10mM EDTA,1%RNA enzyme inhibitor, and 1% aqueous protease.

8. The method according to claim 1, wherein the isothermal amplification in step S2 comprises a recombinase polymerase amplification technique, a recombinase-assisted amplification technique, a loop-mediated isothermal amplification technique, a helicase-dependent isothermal amplification technique, a strand displacement isothermal amplification technique, a nucleic acid sequence-based isothermal amplification technique, a rolling circle amplification, and a nicking endonuclease amplification reaction.

9. The rapid detection method of coronaviruses of non-diagnostic interest as recited in claim 8, wherein said multiple isothermal amplifications of step S2 comprise sequentially performing a single recombinase polymerase amplification and a single nicking endonuclease amplification.

10. The method according to claim 9, wherein the nicking endonuclease used for nicking endonuclease amplification is one or more of nb.btsi, nt.bspqi, nt.cvipii, nt.bstnbi, nb.bsrdi, nb.btsi, nt.alwi, nb.bvci, nt.bbvci, nb.bsmi, nb.bssi, nt.bsmai, and the strand displacing nucleic acid polymerase used for nicking endonuclease amplification is one or more of bst3.0 DNA polymerase, bst2.0 DNA polymerase, klenow DNA polymerase, phi29 DNA polymerase.

11. The method for rapid detection of novel coronaviruses of non-diagnostic interest as recited in claim 10, wherein the basic sequence of the forward primer comprises the nicking site CACTGC and the protecting base of the nicking endonuclease by multiple isothermal amplifications.

12. The rapid detection method of coronavirus of non-diagnostic interest as recited in claim 11, wherein the hybridization product is detected by a colloidal gold test strip.

13. A novel rapid coronavirus detection kit is characterized by comprising magnetic beads, cell lysate, isothermal amplification primers, isothermal amplification enzymes, probes, specific exonucleases and detection test paper; the magnetic beads comprise concanavalin A coated magnetic beads.

14. The kit of claim 13, wherein the probe is a modified oligonucleotide probe that is one or more of biotin-modified, digoxin-modified, texas red-modified, phosphorylated-modified, amino-modified, sulfhydryl-modified, thio-modified, fluorophore-modified, quencher-modified.

15. The kit according to claim 14, wherein the probe has a base sequence of SEQ ID NO.1.

16. The kit of claim 13, wherein the specific exonuclease is one or more of exonuclease VII, lambda exonuclease, T5 exonuclease, T7 exonuclease, exonuclease V, exonuclease III, recJ1 exonuclease, exonuclease I.

17. The kit of claim 13, wherein the cell lysate comprises 10mM HEPES,0.3%SDS,0.5%IGEPAL-CA630,1.5mM MgCl ₂ 0.1% diethyl pyrocarbonate, 10mM KCl,50mM Tris,10mM EDTA,1%RNA enzyme inhibitor, and 1% aqueous protease.

18. The kit of claim 13, wherein the isothermal amplification enzymes comprise a recombinase polymerase and a nicking endonuclease.

19. The kit of claim 18, wherein the basic sequence of the isothermal amplification forward primer comprises the nicking site CACTGC and protecting base of the nicking endonuclease.

20. The kit of claim 13, wherein the test strip comprises a colloidal gold test strip.

21. Use of a kit according to any one of claims 13-20 for the preparation of a reagent for detecting novel coronaviruses and/or for detecting diseases caused by novel coronaviruses.