CN113817871A - Detection method and kit for coronavirus - Google Patents

Abstract

The invention relates to a detection method and a kit for coronavirus, wherein the detection method comprises the following steps: 1) collecting a sample; 2) extracting viral RNA in the sample; 3) performing PCR amplification by using the virus RNA as a template; 4) detecting the PCR amplification product by using a mass spectrometer, and judging the result; wherein, the PCR amplification in step 3) requires the use of primers and probes, which can be used for detecting 7 human coronaviruses, and they are: human coronavirus 229E, NL63, HKU1, OC43, SARS-CoV, MERS-CoV and SARS-CoV-2, wherein the SARS-CoV-2 virus comprises S, L, V, G, GH, GR, GV and GRY typing, UK variant, south Africa variant and Brazilian variant, and the sequences of the primer and the probe are SEQ ID NO.1-SEQ ID NO. 63.

Description

Detection method and kit for coronavirus

Technical Field

The invention belongs to the technical field of molecular biology detection, and particularly relates to a detection method capable of detecting various coronaviruses and provides a kit suitable for the detection method, wherein the various coronaviruses comprise novel coronaviruses SARS-CoV-2 molecular typing and variant strains.

Background

Coronaviruses (CoVs), which belong to the family of coronaviridae, are a large family of viruses that are widely found in nature and that infect humans and animals, and are named when the virion surface is observed under an electron microscope to have coronaries-like fibers. The genome of the coronavirus is large, the single-stranded positive-strand gene sequence of the coronavirus is 26-32 kb in length, and the coronavirus is an RNA virus with the longest natural sequence. The persistent epidemic new Coronavirus pneumonia (Coronavir disease 2019, COVID-19) caused by Severe acute respiratory syndrome Coronavirus type 2 (SARS-CoV-2) initially outbreak in 12 months of 2019, and now poses an unprecedented threat to global public health and economy. SARS-CoV-2 infection mainly causes respiratory infectious diseases mainly including pulmonary diseases, and also causes serious complications such as acute myocardial injury and acute respiratory distress syndrome, and even death. Although the mortality rate of Severe acute respiratory syndrome coronavirus (SARS-CoV) infection appeared in 2003 and Middle east respiratory syndrome coronavirus (MERS-CoV) infection appeared in 2012 was higher than that of SARS-CoV-2, the spread rate of SARS-CoV-2 was much higher than that of MERS-CoV and SARS-CoV. By 17.08.2021, over 200 countries have been affected by the virus, and despite all intervention and control measures, the number of laboratory-diagnosed COVID-19 cases has increased, with over 2 million cases of infection, and over 400 million deaths (: (a)https://gisanddata.maps.arcgis.com/apps/opsdashboard/ index.html#/bda75947440fd40299423467b48e9ecf6)。

In the case of global outbreaks, rapid detection of SARS-CoV-2 infection as early as possible (preferably at the earliest stage of symptoms) is critical to control viral spread, and in particular, the clinical manifestations of SARS-CoV-2 infected patients are very similar to those of other seasonal respiratory tract infected patients such as influenza and are difficult to distinguish.

Therefore, there is an urgent need for a specific and sensitive method for detecting, identifying and screening SARS-CoV-2 in order to determine COVID-19 positive cases at an early stage before clinical symptoms appear in the patient. Currently, SARS-CoV-2 detection is mainly based on two diagnostic strategies: firstly, detecting virus RNA in respiratory tract specimens (nasopharyngeal swab, pharyngeal swab, saliva, sputum and lower respiratory tract secretion) by a molecular diagnosis method; the second is a serological or antibody test that uses a blood sample to detect specific antibodies. Serological tests allow the patient to be monitored for immune response and antibody production during the acute phase (first week of onset) and the convalescent phase (2-4 weeks after onset). Serotype detection is therefore particularly useful for retrospective studies of COVID-19 infection, but not for the identification of early stage SARS-CoV-2 infection. Since SARS-CoV-2 genome sequence information is published for the first time, a series of Nucleic acid amplification methods (NAAT) are rapidly established by virtue of the advantages of short time consumption, simple and convenient operation, automation and the like for detecting SARS-CoV-2 virus RNA, and comprise Next Generation Sequencing (NGS), real-time quantitative reverse transcription polymerase chain reaction (RT-PCR), loop-mediated isothermal amplification (LAMP), Recombinase Polymerase Amplification (RPA) and a method based on a regular interval short palindromic repeat (CRISPR) mechanism. In the early stages of a new outbreak of a coronary epidemic, NGS technology recognizes and discovers the pathogen SARS-CoV-2 in a timely manner, without the knowledge of the causative pathogen. In addition to being used to confirm the suspected COVID-19 disease, NGS technology can also help us to monitor mutations that may occur in the SARS-CoV-2 genome, which is important for understanding the pathogenic mechanism and evolution of the SARS-CoV-2 virus. Although the price of NGS is decreasing year by year, the cost is still high, the whole operation procedure is complicated (including time-consuming and complex library preparation and data processing), professional personnel are required for data analysis, and the disposable sample is limited, which greatly limits the application of NGS in large-scale outbreak. The real-time RT-PCR detection of SARS-CoV-2 is a detection method with strong specificity and high sensitivity, is recommended by the world health organization as an effective and simple method, and is also a gold standard method for identifying COVID-19 pandemic in current research and clinical laboratories. Although real-time RT-PCR assays have become widely used, the increasing demand for assays on a global scale has led to a shortage of reagents and associated consumables, thereby affecting the diagnosis of more and more suspected COVID-19 infections. To meet the unprecedented demand for SARS-CoV-2 diagnosis worldwide, many alternative methods, such as LAMP, RPA and CRISPR, have been developed to achieve equivalent levels of detection of viral RNA. Although these detection methods are faster, simpler, less costly and use less time to confirm the diagnosis of COVID-19 than NGS, they do not provide information on the subtype and mutation of SARS-CoV-2. Therefore, there is an urgent need to develop and establish a comprehensive diagnostic method, which can be used not only for virus identification, but also for typing and mutation detection of SARS-CoV-2 genome, and provide information for epidemic prevention and control in time.

Mass Spectrometry (MS) is a method for qualitative and quantitative analysis and identification of a sample by measuring the mass-to-charge ratio (m/z) of ions of an analyte. The basic working principle of the mass spectrometer is as follows: firstly, molecules of each component in a sample are ionized in an ion source to generate ions (with positive charges or negative charges) with different mass-to-charge ratios; then forming an ion beam by accelerating extraction and transmitting a modulation electric field, and entering a mass analyzer; in the mass analyzer, the ions with different mass-to-charge ratios are focused on an ion detector according to the mass-to-charge ratios by using different acting forces of the electric field and the magnetic field, and finally the mass spectrograms of the ions are obtained by a signal recording system. With the invention of electrospray ionization (ESI) and matrix-assisted laser desorption/ionization (MALDI) ion sources, mass spectrometry has played an increasingly important role in the identification of organic biomolecules. The clinical examination is gradually carried out, and a new technical revolution is brought to the clinical examination. Compared with the traditional immunization and biochemical methods, the clinical medicine mass spectrum has the advantages of high detection speed, strong sample analysis capability, strong platform expansibility and the like, so that the mass spectrum is widely applied to the fields of sequence analysis, genotyping, single nucleotide polymorphism analysis, clinical detection and the like.

Disclosure of Invention

The invention aims to provide a detection method and a corresponding kit which have strong specificity, high sensitivity, rapidness, accuracy and capability of detecting various coronaviruses. The present invention further provides primers and probes related to the detection method of the present invention, which have high specificity.

The detection method of the coronavirus comprises the following steps:

1) collecting a sample;

2) extracting viral RNA in the sample;

3) performing PCR amplification by using the virus RNA as a template;

4) and detecting the PCR amplification product by using a mass spectrometer, and judging the result.

Wherein, the sample in the sample collection in the step 1) is a sample to be detected, and the source of the sample comprises a human nasopharynx swab, saliva, a sputum sample, an alveolar lavage fluid sample, a blood sample, lower respiratory secretions, a stool sample or an environment sample.

Wherein, step 2), extracting viral RNA from the sample can adopt any known extraction method, such as: qiagen kit extraction, Trizol LS organic solvent extraction, and magnetic bead extraction.

Wherein, in the PCR amplification of step 3), primers and probes are required to be used, in order to avoid missing detection, the invention provides the primers and probes capable of detecting a plurality of coronaviruses, and the primers and probes can be used for detecting 7 human coronaviruses, which are: human coronavirus 229E, NL63, HKU1, OC43, SARS-CoV, MERS-CoV, and SARS-CoV-2.

The SARS-CoV-2 virus includes S, L, V, G, GH, GR, GV and GRY typing, UK variant, south Africa variant and Brazilian variant.

The sequences of the primer and the probe are shown in SEQ ID NO.1-SEQ ID NO. 63.

Wherein, the special primer and probe for detecting the human coronavirus 229E are shown in SEQ ID NO.28-SEQ ID NO. 30.

Wherein, the special primer and probe for detecting the human coronavirus NL63 are shown as SEQ ID NO.25-SEQ ID NO. 27.

Wherein, the special primer and probe for detecting the human coronavirus HKU1 are shown as SEQ ID NO.55-SEQ ID NO. 57.

Wherein, the special primer and probe for detecting the human coronavirus OC43 are shown in SEQ ID NO.58-SEQ ID NO. 60.

Wherein, the special primer and probe for detecting severe acute respiratory syndrome coronavirus (SARS-CoV) are shown in SEQ ID NO.52-SEQ ID NO. 54.

Wherein, the special primer and probe for detecting middle east respiratory syndrome coronavirus (MERS-CoV) are shown in SEQ ID NO.40-SEQ ID NO. 42.

Wherein, the special primer and probe for detecting the novel coronavirus (SARS-CoV-2) are shown as SEQ ID NO.13-SEQ ID NO.15 and SEQ ID NO.49-SEQ ID NO. 51.

The special primers and probes for detecting SARS-CoV-2 molecular typing and important variant strains are shown in SEQ ID NO.1-SEQ ID NO.9, SEQ ID NO.16-SEQ ID NO.24, SEQ ID NO.31-SEQ ID NO.39, SEQ ID NO.43-SEQ ID NO.48, and SEQ ID NO.61-SEQ ID NO. 63.

The molecular typing and important variant strains for detecting SARS-CoV-2 mean that SARS-CoV-2 can be accurately classified into S, L, V, G, GH, GR, GV and GRY types, and 3 important variant strains, namely a south African variant strain, a Brazil variant strain and a British variant strain.

The invention relates to 21 target spots, each target spot relates to a set of primer groups, each primer group consists of two primers, one is a forward primer, and the other is a reverse primer. Therefore, the invention has 21 sets of primer groups and probes, and realizes multiple detection reactions aiming at 21 detection targets respectively.

The correspondence between 21 targets and primers and probes is as follows:

primer names and sequence information are shown in the following table.

TABLE 1 primer and Probe sequence information

The PCR amplification in step 3) of the invention adopts a one-step multiplex PCR technology to realize multiplex detection reaction, and the basic method is as follows:

after reverse transcription of the extracted RNA sample by RNA reverse transcriptase, PCR amplification of the reverse transcription product is performed by using Taq enzyme. The PCR product was treated with Shrimp Alkaline Phosphate (SAP) and sample spotting was performed after single base extension reaction.

Preferably, the method is as follows:

the extracted virus RNA is detected by a one-step method by using a mixed reaction system containing RNA reverse transcriptase and Taq enzyme, wherein the mixed reaction system contains 7 types of human coronavirus, novel coronavirus types and variant strains. And in accordance with the processing method, the amplified PCR product is processed by SAP, and then sample spotting is carried out after single base extension reaction.

Most preferably, the method is as follows:

viral RNA was extracted from the samples according to the QIAamp Viral RNA Mini kit instructions, while RNase inhibitor was added to the RNA storage tubes. (the above steps may be performed using other nucleic acid extraction kits or methods to accomplish viral RNA extraction). Multiplex PCR reaction: using the viral RNA samples obtained in the above procedure as templates, 10. mu.L of multiplex PCR reaction system was added including: 2.6 μ L of each primer was optimally amplifiedConcentration-increasing Mixed System, 5. mu.L of 2 × Reaction Mix, 0.4. mu.L

III RT/

Taq Mix, 2. mu.L viral RNA sample template. Positive control and negative control reaction tubes were introduced simultaneously for each detection reaction. The conditions of the amplification reaction are as follows: incubating for 30 minutes at 55 ℃ to complete RNA reverse transcription PCR reaction; after incubation at 94 ℃ for 2 minutes, denaturation at 94 ℃ for 15 seconds, annealing at 56.5 ℃ for 30 seconds, extension at 68 ℃ for 1 minute, and after completion of 45 cycles of amplification reaction, additional extension at 68 ℃ for 5 minutes, and then storage at low temperature of 4 ℃. The reaction product was collected and 2 μ L of SAP reaction system was added: 1 u LSAP buffer and 1 u LSAP enzyme. The method is carried out according to the following reaction conditions: SAP was inactivated at 37 ℃ for 15 minutes and at 85 ℃ for 5 minutes. The reaction product was collected and 2. mu.L of a single base extension reaction system was added: 0.4. mu.L of single-base extension buffer, 0.94. mu.L of probe mixture, and 0.041. mu.L of single-base extension enzyme. And (3) amplification reaction conditions: after pre-denaturation at 95 ℃ for 30 seconds, reaction at 94 ℃ for 5 seconds, reaction at 52 ℃ for 5 seconds, reaction at 80 ℃ for 5 seconds, and 5 cycles of the above 2 reactions, the above 3 reactions were incubated at 72 ℃ for 3 minutes after completion of 45 amplification cycles in total. Mixing 11 μ L of non-nucleic acid water with the reaction system, transferring a proper amount of resin to the reaction system, uniformly blowing and sucking for several times, placing on a turnover shaking table, turning at 80rpm for 30 minutes to ensure that the resin is fully contacted with the reaction, and adsorbing cations in the reaction solution.

Reaction product spotting: reaction product spotting: preparing a matrix for nucleic acid mass spectrum spotting: preparing 3-hydroxy-2-picolinic acid (3-hydroxypicolinic acid, 3HPA) into 50mg/mL by using an acetonitrile solution; diammonium citrate (ACD) was formulated to a final concentration of 250 mM/mL; finally, 3 HPA: ACD is prepared into a finally used nucleic acid mass spectrum spotting matrix solution according to the volume ratio of 10: 1. Coating a substrate: about 1. mu.L of the matrix solution was coated in the center of the mass spectrometric detection target plate and left to stand at room temperature for about 2 minutes. Nucleic acid sample spotting: mu.L of the reaction product obtained in (3) was collected, spotted onto a substrate-coated target plate, and after standing at room temperature for about 2 minutes, it was ready for detection using a mass spectrometer.

Step 4) of the invention, the PCR amplification product is detected by a mass spectrometer, and the result is judged, wherein the detection method comprises the following steps:

the PCR reaction product obtained by the treatment is fully mixed and stood with the nucleic acid mass spectrum sample application matrix. Detecting the ionization of molecules of each component in the sample in an ion source by using a mass spectrometer to generate examples with different mass-to-charge ratios; analyzing the electron beam formed by the accelerated extraction and transmission modulation electric field by a mass analyzer of the mass spectrometer; the ions with different mass-to-charge ratios are focused on an ion detector according to the mass-to-charge ratio by utilizing different actions of the electric field and the magnetic field, and a mass spectrogram of the ions is obtained by a signal recording system of a mass spectrometer. Preferably, the method is as follows:

and detecting the processed nucleic acid sample by using an AXIMA mass spectrometer, and analyzing a mass spectrum detection result by using instrument matching software.

Most preferably, the method is as follows:

and (3) detecting the virus RNA nucleic acid sample after the multiplex PCR reaction, the SAP reaction and the single base extension reaction and the resin purification by using an AXIMA mass spectrometer. Simultaneously, analyzing a detection result by using MALDI-MS software designed by the invention; if the detection result is positive, a specific product peak appears at the charge-to-mass ratio of the positive product of the corresponding target gene; if the detection result is negative, a specific product peak appears at the initial charge-to-mass ratio of the corresponding target gene; and (3) integrating the detection conditions of the positive specific product peaks of all targets, judging the detection condition of the human coronavirus of the sample, and determining the molecular type and the site mutation condition of SARS-CoV-2. The invention further provides a kit for detecting coronavirus on the basis of the detection method.

The kit of the present invention can detect 7 kinds of human coronavirus and SARS-CoV-2 virus typing and variant. The kit comprises the primer and the probe.

The kit of the invention can also comprise detection tools and reagents according to requirements, such as reagents required by PCR (polymerase chain reaction) such as solvents, enzymes and reference substances, wherein the tools can comprise use instructions, cotton swabs, brackets, reagent bottles, test paper and other necessary matching products in detection work, and specifically comprise: reacting the buffer mixture, RNA reverse transcriptase with polymerase, Shrimp Alkaline Phosphatase (SAP) components (SAP buffer and SAP enzyme), single base extension reaction components (single base extension buffer, single base extension enzyme);

the kit comprises a positive control and a negative control, wherein the positive control is a positive sample containing each detection target point or a wild type or mutant type positive sample containing the detection target points, and the negative control is a normal human RNA nucleic acid sample.

In the kit, the primers and the probes comprise primers and probes for detecting human coronavirus 229E, and the sequences of the primers and the probes are SEQ ID NO.28-SEQ ID NO. 30.

The kit also comprises a primer and a probe for detecting the human coronavirus NL63, and the sequence of the primer and the probe is SEQ ID NO.25-SEQ ID NO. 27.

The kit also comprises a primer and a probe for detecting the human coronavirus HKU1, and the sequence of the primer and the probe is shown as SEQ ID NO.55-SEQ ID NO. 57.

The kit also comprises a primer and a probe for detecting the human coronavirus OC43, and the sequence of the primer and the probe is shown in SEQ ID NO.58-SEQ ID NO. 60.

The kit also comprises a primer and a probe for detecting the severe acute respiratory syndrome coronavirus (SARS-CoV) of the human coronavirus, and the sequence of the primer and the probe is shown as SEQ ID NO.52-SEQ ID NO. 54.

The kit also comprises a primer and a probe for detecting middle east respiratory syndrome coronavirus (MERS-CoV), and the sequence of the primer and the probe is shown in SEQ ID NO.40-SEQ ID NO. 42.

The kit also comprises a primer and a probe for detecting SARS-CoV-2 virus, and the sequences of the primer and the probe are shown in SEQ ID NO.13-SEQ ID NO.15 and SEQ ID NO.49-SEQ ID NO. 51.

The kit also comprises primers and probes for detecting SARS-CoV-2 virus molecular typing and variant strains, and the sequences of the primers and probes are shown in SEQ ID NO.1-SEQ ID NO.9, SEQ ID NO.16-SEQ ID NO.24, SEQ ID NO.31-SEQ ID NO.39, SEQ ID NO.43-SEQ ID NO.48, and SEQ ID NO.61-SEQ ID NO. 63.

The kit comprises primers and probes shown in SEQ ID NO.1-SEQ ID NO. 63.

The primers, the probes, the reagents and the tools in the kit can meet the requirement of detecting the dosage of a sample for 1 person, and also can meet the dosage of a sample for multiple persons, such as 5 persons, 10 persons, 20 persons, 50 persons, 100 persons, 1000 persons and the like, so as to meet the screening requirement to determine the specific dosage.

The dosage of the sample of 1 person is taken as an example, wherein:

the primers and probes shown in SEQ ID NO.1-SEQ ID NO.63 have the following respective dosage and preparation method:

1) preparing a primer: diluting each primer to 50 mu M, mixing 10 mu L of the primers with the concentrations, and filling the mixture to 1mL by using non-nucleic acid water, namely the primer mixture used in the multiplex PCR reaction used by the sample for 1 person, wherein the dosage of each person is 2.6 mu L.

2) Preparing an extension probe: each probe was diluted to 250. mu.M and the volume of each probe added was calculated as follows: v ═ 9/0.94 × 4 (InM-7.82), where M is the molecular weight of the probe and V is the calculated theoretical amount added per probe in μ L. For probes with molecular weight less than 6000 daltons, mixing according to theoretical addition; probes with molecular weight greater than 6000 daltons were mixed at 1.5 times the theoretical addition. The mixed probe is filled to 1mL by using nuclease-free water, namely the probe mixed solution used for the single-base extension reaction used by the sample for 1 person is obtained, and the dosage of each person is 0.94 mu L.

The invention further comprises the application of the kit, and the application comprises the following steps:

patient respiratory specimens (nasopharyngeal swabs, pharyngeal swabs, saliva, sputum, and lower respiratory secretions) were collected using a viral collection tube. Viral RNA was extracted from the samples according to the QIAamp Viral RNA Mini kit protocol, eluting at 50. mu.L to the recommended kit volume, while RNase inhibitor was added to the RNA storage tubes. (the above steps may be performed using other nucleic acid extraction kits or methods to accomplish viral RNA extraction).

Multiplex PCR reaction: using the viral RNA samples obtained in the above procedure as templates, 10. mu.L of multiplex PCR reaction system was added including: 2.6. mu.L of the mixed system of the optimal amplification concentrations of the respective primers, 5. mu.L of 2 × Reaction Mix, 0.4. mu.L

III RT/

Taq Mix, 2. mu.L viral RNA sample template. Positive control and negative control reaction tubes were introduced simultaneously for each detection reaction. The conditions of the amplification reaction are as follows: incubating for 30 minutes at 55 ℃ to complete RNA reverse transcription PCR reaction; after incubation at 94 ℃ for 2 minutes, denaturation at 94 ℃ for 15 seconds, annealing at 56.5 ℃ for 30 seconds, extension at 68 ℃ for 1 minute, and after completion of 45 cycles of amplification reaction, additional extension at 68 ℃ for 5 minutes, and then storage at low temperature of 4 ℃. The reaction product was collected and 2 μ L of SAP reaction system was added: 1 u LSAP buffer and 1 u LSAP enzyme. The method is carried out according to the following reaction conditions: SAP was inactivated at 37 ℃ for 15 minutes and at 85 ℃ for 5 minutes. The reaction product was collected and 2. mu.L of a single base extension reaction system was added: 0.4. mu.L of single-base extension buffer, 0.94. mu.L of probe mixture, and 0.041. mu.L of single-base extension enzyme. And (3) amplification reaction conditions: after pre-denaturation at 95 ℃ for 30 seconds, reaction at 94 ℃ for 5 seconds, reaction at 52 ℃ for 5 seconds, reaction at 80 ℃ for 5 seconds, and 5 cycles of the above 2 reactions, the above 3 reactions were incubated at 72 ℃ for 3 minutes after completion of 45 amplification cycles in total. Mixing 11 μ L of non-nucleic acid water with the reaction system, transferring a proper amount of resin to the reaction system, uniformly blowing and sucking for several times, placing on a turnover shaking table, turning at 80rpm for 30 minutes to ensure that the resin is fully contacted with the reaction, and adsorbing cations in the reaction solution. Reaction product spotting: reaction product spotting:preparing a matrix for nucleic acid mass spectrum spotting: using acetonitrile solution3-hydroxy-2-pyridinecarboxylic acid(3-hydroxypicolinic acid, 3HPA) is prepared into 50 mg/mL; diammonium citrate (ACD) was formulated to a final concentration of 250 mM/mL; finally, 3 HPA: ACD is prepared into a finally used nucleic acid mass spectrum spotting matrix solution according to the volume ratio of 10: 1. Coating a substrate: about 1. mu.L of the matrix solution was coated in the center of the mass spectrometric detection target plate and left to stand at room temperature for about 2 minutes. Nucleic acid sample spotting: mu.L of the reaction product obtained in (3) was collected, spotted onto a substrate-coated target plate, and after standing at room temperature for about 2 minutes, it was ready for detection using a mass spectrometer.

Mass spectrum detection: the reaction product was detected using an AXIMA mass spectrometer, and the detection results were analyzed using MALDI-MS software.

And (4) interpretation of results: and (5) judging the detection result. If the detection result is positive, a specific product peak appears at the positive product charge-to-mass ratio of the corresponding target gene (the positive product charge-to-mass ratio is shown in table 2); if the detection result is negative, a specific product peak appears at the initial charge-to-mass ratio of the corresponding target gene (the charge-to-mass ratio of the initial product is shown in Table 2). And (3) integrating the detection conditions of the positive specific product peaks of all targets, judging the detection condition of the human coronavirus of the sample, and determining the molecular type and the site mutation condition of SARS-CoV-2.

TABLE 2 initial and Positive Charge-to-mass ratios of the detection targets

Numbering	Detection target	Initial mass to charge ratio (M/Z)	Positive mass to charge ratio (M/Z)
				1	11083	7272.6	7559.8
2	22227	4878.8	5126.6
				3	23012	5833.6	6120.6
4	23063	4486.9	4760.8
				5	23403	5473.2	5800.5
6	241	5762.4	6009.5
				7	25563	6891.08	7178.6
8	26144	6687.9	6935.53
				9	28144	7134.4	7405.75
10	28882	4297.6	4584.63
				11	3037	6053.69	6340.63
12	8782	6754.9	7002.4
				13	COVID-19_N	5089.8	5361.1
14	COVID-19_RdRp	7494.5	7822.2
				15	HCoV-229E	6277.6	6525.3
16	HCoV-HKU1	7926.8	8198.4
				17	MERS-CoV	7038.2	7365.7
18	HCoV-OC43	8342.4	8670.5
				19	SARS-CoV	7696.1	8023.1
20	HCoV-NL63	6155.8	6443.2
				21	RNase_P	4921.8	5169.2

The invention further provides an analysis method for simultaneously displaying the mass spectrum detection peak images of a plurality of samples, which comprises MALDI-MS mass spectrum result analysis software of the invention, can simultaneously present the types of 7 human coronaviruses and SARS-CoV-2 in different samples and the detection conditions of variant strains, and is convenient for simultaneously presenting the analysis results of a plurality of samples (figure 1).

The MALDI-MS mass spectrometry software comprises the following calculation modules:

1. analyzing the mass spectrum detection result by using MALDI-MS (matrix-assisted laser Desorption-Mass Spectrometry) matched with an AXIMA mass spectrum instrument;

2. selecting results to be displayed (10 mass spectrum detection results can be displayed simultaneously) by using a Load Data module in software;

3. and (3) using a Display contents program in result analysis to perform unified processing on the displayed result, setting the program as a Process, a Stack and a Set 1-5, and realizing fitting analysis of the same coordinate of a plurality of samples.

The collective application of these modules enables comparative analysis of multiple samples fitted to the same coordinate for comparison of the sample to a negative control or other sample (fig. 2).

The primer and probe of the invention are screened, designed, synthesized, applied and finally determined after obtaining good effect, and the primer and probe are selected as the target gene (including ORF1a gene and N gene of SARS-CoV-2 and RdRp gene of another 6 kinds of human coronavirus) for 7 kinds of human coronavirus detection, and the related mutation site region (UK variant, south African variant and Barwest variant) capable of accurately dividing SARS-CoV-2 into S, L, V, G, GH, GR, GV and GRY types and identifying the important variant of new type coronavirus is selectedhttps://www.gisaid.org/references/ statements-clarifications/clade-and-lineage-nomenclature-aids-in-genomic- epidemiology-of-active-hcov-19-viruses/)

The primer is used as a detection target for molecular typing and variant identification of SARS-CoV-2 virus, and simultaneously, a human RNase P gene is selected as an internal control for monitoring the extraction condition of sample nucleic acid. Firstly, 7 kinds of human coronavirus which are completely annotated are downloaded from GenBank database (https:// www.ncbi.nlm.nih.gov/GenBank) and GISAID database (https:// www.gisaid.org) as representative strain gene sequences of reference sequences, the nucleic acid sequences BLAST (https:// blast.ncbi.nlm.nih.gov/blast.cgi) is carried out on the reference sequences and the nr database of NCBI, and the obtained results are downloaded and aligned to obtain more detection target gene sequences. And (3) designing specific amplification primers and probes for the conserved region sequences of the target genes obtained by analysis by using primer design software, wherein the design result must meet each target gene, and the penalty of each standard of the detection reagent cannot exceed the condition of the set threshold value of the software. If the condition is not met, the conserved region of the target gene with the undesirable design needs to be selected again according to the software prompt. After many trials, the optimal primer and probe for accurately detecting 7 human coronaviruses and the mutation site for SARS-CoV-2 typing are finally obtained. The reagents were evaluated using the online Primer validation tool Primer-BLAST (https:// www.ncbi.nlm.nih.gov/tools/Primer BLAST /) supplied by NCBI to validate the specificity of the primers.

Designing a primer and a probe: and (3) designing specific amplification primers and probes for the conserved region sequences of the target genes obtained by analysis by using primer design software, wherein the design result must meet each target gene, and the penalty of each standard of the detection reagent cannot exceed the condition of the set threshold value of the software. If the condition is not met, the conserved region of the target gene with the undesirable design needs to be selected again according to the software prompt. After many trials, the optimal primer and probe for accurately detecting 7 human coronaviruses and the mutation site for SARS-CoV-2 typing are finally obtained.

Determination of the multiplex detection primer group and the probe: the reagents were evaluated using the online Primer validation tool Primer-BLAST (https:// www.ncbi.nlm.nih.gov/tools/Primer BLAST /) supplied by NCBI to validate the specificity of the primers. The optimal primer pair which can accurately identify 7 human coronaviruses and distinguish the wild type and the mutant type of SARS-CoV-2 is selected to be combined with the probe to form a final mass spectrometry system with 21 weight per hole.

The primer and probe of the present invention, whose sequences are completely novel, are designed according to the above-described method, and can be prepared, for example, by a method of artificially chemically synthesizing Oligo DNA or a method of chemically synthesizing Oligo DNA using β -acetonitrile phosphoramidite.

These preparation methods are conventional per se and reference is made to textbooks of molecular cloning protocols.

The invention isThe 21 specific detection targets include: taking interspecific specific, intraspecies conserved regions (RdRp gene) as target genes for detection of 6 coronaviruses of human coronavirus 229E, NL63, HKU1, OC43, severe acute respiratory syndrome coronavirus (SARS-CoV), and middle east respiratory syndrome coronavirus (MERS-CoV); interspecific specific, interspecific conserved genes or regions (including ORF1a gene and N gene) as target genes for SARS-CoV-2 virus detection; the 12 different targeting regions cover T8782G, C28144T, G11083T, G26144T, C241T, C3037T, A23043G, G28882A, G25563T, A23063T, G23012A and C22227T 12 important mutation sites for molecular typing of SARS-CoV-2 and detection of important mutant strains, so that accurate classification of 8 types of SARS-CoV-2 virus S, L, V, G, GH, GV, GR and GRY and detection of 3 important variants (British variant, south African variant and Brazilian variant) are realized; the human RNase P gene is included in the detection system as an internal reference control for monitoring the extraction condition of the sample nucleic acid. The invention selects interspecific specific and intraspecies conserved genes or regions as target genes (including ORF1a gene and N gene of SARS-CoV-2 and RdRp gene of another 6 kinds of human coronavirus) for detection of 7 kinds of human coronavirus, selects related mutation site region (including S, L, V, G, GH, GV, GR and GRY types) capable of accurately dividing SARS-CoV-2 into S, L, V, G, GH, GV, GR and GRY types, and identifies important variant strains (British variant strain, south African variant strain and Barre variant strain) of novel coronavirushttps://www.gisaid.org/references/statements-clarifications/clade-and- lineage-nomenclature-aids-in-genomic-epidemiology-of-active-hcov-19-viruses/)

The PCR product is used as a detection target for molecular typing of SARS-CoV-2 virus, and simultaneously, a human RNase P gene is selected as an internal control for monitoring the extraction condition of sample nucleic acid.

The method of the present invention can detect 7 kinds of human coronavirus from clinical sample fast, specifically and sensitively, and perform molecular typing and important variant detection on SARS-CoV-2 virus. Compared with other existing technologies for detecting human coronavirus and SARS-CoV-2, the scheme of the invention has the following advantages:

firstly, the method improves and optimizes the whole detection reaction for the first time, and uses the one-step multiplex PCR technology and the nucleic acid mass spectrometry detection analysis for combination, so as to realize direct detection and typing after the nucleic acid of the clinical sample is extracted, and the RNA reverse transcription is not required to be additionally carried out, thereby greatly shortening the detection time and reducing the consumption of reagents; the SAP is used for treating a PCR reaction product, and residual dNTP is digested, so that compared with the conventional reagent, the reaction time is saved, and the reaction cost is reduced; secondly, compared with the traditional PCR or other molecular detection technologies, the mass spectrometric identification is based on the molecular weight of the analyte, and the mass of the extension probe can be distinguished by reasonably selecting and designing and properly adding modified bases, so that the aim of detecting multiple target genes at one time is fulfilled, and the target gene detection efficiency is effectively improved; the method can combine and match the detection targets according to the requirements, flexibly increase and decrease the design scheme of the reagent according to the requirements, and provides great convenience for the subsequent detection of other variant strains of SARS-CoV-2 virus; MALDI-MS software is used for analyzing the mass spectrum detection result, the presentation of the detection results of a plurality of nucleic acid samples and the analysis and comparison of the same coordinate are realized in a breakthrough manner, and convenience is brought to the comparison of the detection results among the samples.

The terms appearing herein are further explained and illustrated below:

primer: two-segment oligonucleotide sequence for amplifying target gene

And (3) probe: nucleic acid sequence for target gene detection in single base extension reaction

And (3) PCR amplification: polymerase chain reaction, a molecular technique for amplifying specific DNA fragments

One-step multiplex PCR technique: a reaction system comprising RNA reverse transcriptase and DNA polymerase, and a technique for simultaneously performing reverse transcription of RNA and multiplex PCR amplification reaction in one reaction

RNA reverse transcription, namely synthesizing DNA by taking RNA as a template, treating a PCR reaction product by using a synthesized DNA chain as DNA SAP complementary to the RNA, digesting the residual dNTP: the shrimp alkaline phosphatase is used for eliminating redundant mononucleotide in a one-step multiple PCR reaction system, so that the subsequent reaction is prevented from being influenced

The quality of the extension probe can be distinguished by adding a modified base to the amplification primer (ACGTTGGATG) to avoid interference of the primer with the analysis of the detection result

Rpm: rotational speed

PCR polymerase chain reaction

SAP shrimp alkaline phosphatase

Amplifying buffer: one-step amplification reaction buffer solution

Reaction Mix: reaction premix

QIAamp Viral RNA Mini kit: viral RNA extraction kit

III RT/

Taq Mix: reverse transcriptase

dNTP: oligonucleotide for one-step amplification reaction

Single base extension buffer: buffer solution for single-base extension reaction

Description of the drawings:

FIG. 1 shows the results of typing 7 human coronaviruses and SARS-CoV-2 molecules in a plurality of samples and detecting a mutant.

FIG. 2 shows the results of 7 human coronavirus and SARS-CoV-2 molecular typing and variant detection in the same coordinate. Note: red: RNA nucleic acid samples containing the mutation sites of SARS-CoV-2 virus strains; blue color: normal human RNA nucleic acid samples.

Detailed Description

The invention is further illustrated by the following examples. The embodiments are implemented on the premise of the invention, and give detailed implementation modes and specific operation processes. The specific embodiments and operations described herein are merely illustrative of the present invention, and the scope of the present invention is not limited to the following examples. The following embodiments and specific procedures of examples of 7 human coronavirus detection and SARS-CoV-2 virus molecular typing and variant detection using the present invention with respect to clinical nucleic acid samples are described below.

Example 1, test case:

collecting samples:

patient respiratory specimens (nasopharyngeal swabs, pharyngeal swabs, saliva, sputum, and lower respiratory secretions) were collected using a dedicated viral collection tube.

RNA extraction:

viral RNA was extracted from the samples according to the QIAamp Viral RNA Mini kit protocol, eluting at 50. mu.L to the recommended kit volume, while RNase inhibitor was added to the RNA storage tubes. (the above steps can be performed by other nucleic acid extraction kits or methods)

Multiplex PCR reaction (Using the kit of example 2 of the present invention), the viral RNA sample obtained in the above procedure was used as a template, and 10. mu.L of a multiplex PCR reaction system was added, including: 2.6. mu.L of the mixed system of the optimal amplification concentrations of the respective primers, 5. mu.L of 2 × Reaction Mix, 0.4. mu.L

III RT/

Taq Mix, 2. mu.L viral RNA sample template. Positive control and negative control reaction tubes were introduced simultaneously for each detection reaction. The conditions of the amplification reaction are as follows: incubating for 30 minutes at 55 ℃ to complete RNA reverse transcription PCR reaction; after incubation at 94 ℃ for 2 minutes, denaturation at 94 ℃ for 15 seconds, annealing at 56.5 ℃ for 30 seconds, and extension at 68 ℃ for 1 minute, 45 cycles of amplification reaction were completed, and after additional extension at 68 ℃ for 5 minutes, the cells were stored at low temperature to 4 ℃. The reaction product was collected and 2 μ L of SAP reaction system was added: 1 u LSAP buffer and 1 u LSAP enzyme. The method is carried out according to the following reaction conditions: SAP was inactivated at 37 ℃ for 15 minutes and at 85 ℃ for 5 minutes. The reaction product was collected and 2. mu.L of a single base extension reaction system was added: 0.4. mu.L of single-base extension buffer, 0.94. mu.L of probe mixture, and 0.041. mu.L of single-base extension enzyme. And (3) amplification reaction conditions: after pre-denaturation at 95 ℃ for 30 seconds, reaction at 94 ℃ for 5 seconds, reaction at 52 ℃ for 5 seconds, reaction at 80 ℃ for 5 seconds, and 5 cycles of the above 2 reactions, the above 3 reactions were incubated at 72 ℃ for 3 minutes after completion of 45 amplification cycles in total.Mixing 11 μ L of non-nucleic acid water with the reaction system, transferring a proper amount of resin to the reaction system, uniformly blowing and sucking for several times, placing on a turnover shaking table, turning at 80rpm for 30 minutes to ensure that the resin is fully contacted with the reaction, and adsorbing cations in the reaction solution.

Reaction product spotting: preparing a matrix for nucleic acid mass spectrum spotting: using acetonitrile solution3-hydroxy-2-pyridinecarboxylic acid(3-hydroxypicolinic acid, 3HPA) is prepared into 50 mg/mL; diammonium citrate (ACD) was formulated to a final concentration of 250 mM/mL; finally, 3 HPA: ACD is prepared into a finally used nucleic acid mass spectrum spotting matrix solution according to the volume ratio of 10: 1. Coating a substrate: about 1. mu.L of the matrix solution was coated in the center of the mass spectrometric detection target plate and left to stand at room temperature for about 2 minutes. Nucleic acid sample spotting: mu.L of the reaction product obtained in (3) was collected, spotted onto a substrate-coated target plate, and after standing at room temperature for about 2 minutes, it was ready for detection using a mass spectrometer.

Mass spectrum detection: the reaction product was detected using an AXIMA mass spectrometer, and the detection results were analyzed using MALDI-MS software.

And (4) interpretation of results: and (5) judging the detection result. If the detection result is positive, a specific product peak appears at the positive product charge-to-mass ratio of the corresponding target gene (the positive product charge-to-mass ratio is shown in table 2); if the detection result is negative, a specific product peak appears at the initial charge-to-mass ratio of the corresponding target gene (the charge-to-mass ratio of the initial product is shown in Table 2). And (3) integrating the detection conditions of the positive specific product peaks of the targets, judging the detection conditions of the human coronavirus in the sample, and determining the molecular type and the site mutation condition of SARS-CoV-2 (as shown in figure 2).

Wherein the final concentration of the primers and the probes in the multiplex PCR reaction system is 50 mu M of the final concentration of each primer, and the final concentration of each probe is 250 mu M.

In the case of the example 2, the following examples are given,

the kit comprises the following components:

1. primer and probe mixed solution: each of 21 primers was diluted to 50. mu.M, and 10. mu.L of each primer was mixed at the above concentration and supplemented to 1mL with a non-nucleic acid solution. Each probe was diluted to 250 μ M and each probe was according to the formula: the theoretical amount of each probe added was calculated as (InM-7.82) × 9/0.94 × 4 in μ L and M is the molecular mass in dalton. Wherein the probes with the molecular weight less than 6000 daltons are mixed according to the theoretical addition amount; probes with molecular weight greater than 6000 daltons were mixed at 1.5 times the theoretical addition and made up to 1mL using non-nucleic acid water. The mixed primers and probes are used in multiplex PCR and single-base extension reactions, respectively.

2. The reaction system comprises the following components: the one-step multiplex PCR Reaction system comprises 2 × Reaction Mix,

III RT/

taq Mix, PCR primer mixture, and non-nucleic acid water; the SAP reaction system comprises: SAP buffer and SAP enzyme; the single base extension reaction comprises: single base extension buffer, probe mixture, single base extension reaction enzyme, and no nucleic acid water. The extracted viral RNA was subjected to the above-mentioned reaction, and then resin-purified.

3. Nucleic acid spotting matrices: 50mg/mL of3-hydroxy-2-pyridinecarboxylic acid(3-Hydroxypicolinic acid, 3HPA) with 250mM/mL hydrogencitrate diamine (ACD) as 10:1, the nucleic acid matrix is prepared. The extracted nucleic acid is subjected to the PCR reaction treatment and resin purification, and then is fully mixed with the prepared nucleic acid matrix for detection by using a mass spectrometer.

Examples 3,

Performance testing of the kit

1. Accuracy verification

The results of selecting RNA extracted from 5 SARS-CoV-2 isolates infected with cells and 2 positive patient clinical specimens, and testing by referring to the method and procedure of example 1 above show that these samples can accurately identify two SARS-CoV-2 target genes, and determine that these samples are SARS-CoV-2 positive; and the samples are classified into different types according to the detection results of 12 mutation sites of the same hole for detecting SARS-CoV-2 typing and variant strains. And sequencing the 12 marked mutation sites of the samples by using a PCR-sequencing method, wherein the result shows that the typing result of the method is consistent with the typing result obtained by the PCR-sequencing method. In conclusion, the method has good detection and typing performance.

2. Specificity verification

The method was evaluated for specificity by selecting pathogens common to respiratory infections, including adenovirus, enterovirus, bocavirus, metapneumovirus type A and B, rhinovirus, influenza A H1N1, influenza A H3N2, influenza B, parainfluenza viruses (types 1 to 4), respiratory syncytial virus (types A and B), Legionella pneumophila, Bordetella pertussis, Mycoplasma pneumoniae, Chlamydia pneumoniae, Haemophilus influenzae, Staphylococcus aureus, Moraxella catarrhalis, Klebsiella pneumoniae, Pseudomonas aeruginosa, Acinetobacter baumannii, Escherichia coli, Streptococcus pneumoniae, and Neisseria meningitidis, and testing was performed with reference to the method and procedure of example 1 above, and the results showed that, the 21-fold detection primer group and the probe contained in the method have no cross reaction to all tested pathogens, and the method is good in specificity.

3. Sensitivity detection

The sensitivity was evaluated using a multiple dilution of gradient positive plasmids, tested according to the method and procedure of example 1 above, and repeated 3 times for each dilution, showing that all targets detected by this method were stably detected at 10 copies/reaction.

Sequence listing

<110> institute of pathogenic biology of Chinese academy of medical sciences

<120> a novel coronavirus SARS-CoV-2 detection and molecular typing method and kit

<130>

<160> 4

<210> 1

<211> 30

<212> RNA

<213> SARS-CoV-2_N-28882

<400> 1

acgttggatg caaagcaaga gcagcatcac 30

<210> 2

<211> 35

<212> RNA

<213> SARS-CoV-2_N-28882

<400> 2

Acgttggatg ttcgcccaca tgagggacaa ggaca 35

<210> 3

<211> 14

<212> RNA

<213> SARS-CoV-2_N-28882

<400> 3

Ccaggcagca gtag 14

<210> 4

<211> 30

<212> RNA

<213> SARS-CoV-2_S-23063

<400> 1

Acgttggatg acagttgctg gtgcatgtag 30

<210> 5

<211> 31

<212> RNA

<213> SARS-CoV-2_S-23063

<400> 2

acgttggatg ctttacaatc atatggtttc c 31

<210> 6

<211> 15

<212> RNA

<213> SARS-CoV-2_S-23063

<400> 3

Ggtttccaac ccact 15

<210> 7

<211> 30

<212> RNA

<213> SARS-CoV-2_S-22227

<400> 1

Acgttggatg tacctattgg caaatctacc 30

<210> 8

<211> 30

<212> RNA

<213> SARS-CoV-2_S-22227

<400> 2

Acgttggatg tttagtgcgt gatctccctc 30

<210> 9

<211> 16

<212> RNA

<213> SARS-CoV-2_S-22227

<400> 3

Cctcagggtt tttcgg 16

<210> 10

<211> 30

<212> RNA

<213> RNase_P

<400> 1

Acgttggatg tgagcggctg tctccacaag 30

<210> 11

<211> 30

<212> RNA

<213> RNase_P

<400> 2

Acgttggatg tggcggtgtt tgcagatttg 30

<210> 12

<211> 16

<212> RNA

<213> SARS-CoV-2_ORF1a

<400> 3

Cagagcgggt tctgac 16

<210> 13

<211> 30

<212> RNA

<213>

<400> 1

Acgttggatg aattggaacg ccttgtcctc 30

<210> 14

<211> 30

<212> RNA

<213> SARS-CoV-2_ORF1a

<400> 2

Acgttggatg ttggttcacc gctctcactc 30

<210> 15

<211> 17

<212> RNA

<213> SARS-CoV-2_ORF1a

<400> 3

Ctctcactca acatggc 17

<210> 16

<211> 30

<212> RNA

<213> SARS-CoV-2_S-23403

<400> 1

Acgttggatg ttctaaccag gttgctgttc 30

<210> 17

<211> 30

<212> RNA

<213> SARS-CoV-2_S-23403

<400> 2

Acgttggatg acacgccaag taggagtaag 30

<210> 18

<211> 18

<212> RNA

<213> SARS-CoV-2_S-23403

<400> 3

Acttctgtgc agttaaca 18

<210> 19

<211> 30

<212> RNA

<213> SARS-CoV-2_5'UTR-241

<400> 1

Acgttggatg ccatcttacc tttcggtcac 19

<210> 20

<211> 30

<212> RNA

<213> SARS-CoV-2_5'UTR-241

<400> 2

Acgttggatg actcgtctat cttctgcagg 30

<210> 21

<211> 19

<212> RNA

<213> SARS-CoV-2_5'UTR-241

<400> 3

Catcagcaca tctaggttt 19

<210> 22

<211> 30

<212> RNA

<213> SARS-CoV-2_ORF1ab-3037

<400> 1

Acgttggatg ggagtatggc tacatactac 30

<210> 23

<211> 30

<212> RNA

<213> SARS-CoV-2_ORF1ab-3037

<400> 2

Acgttggatg tcttcttcat cctcatctgg 30

<210> 24

<211> 20

<212> RNA

<213> SARS-CoV-2_ORF1ab-3037

<400> 3

Cccccctcat ctggagggta 20

<210> 25

<211> 30

<212> RNA

<213> HCoV-NL63

<400> 1

Acgttggatg ctgaacttgt ttatgagaac 30

<210> 26

<211> 30

<212> RNA

<213> HCoV-NL63

<400> 2

Acgttggatg tgtcaacctg tacattaccc 30

<210> 27

<211> 20

<212> RNA

<213> HCoV-NL63

<400> 3

Ttactagcag gtttaacagg 20

<210> 28

<211> 29

<212> RNA

<213> HCoV-229E

<400> 1

Acgttggatg tctgttttta catgcttcc 29

<210> 29

<211> 30

<212> RNA

<213> HCoV-229E

<400> 2

Acgttggatg taagtaaccg tatcagaacc 30

<210> 30

<211> 21

<212> RNA

<213> HCoV-229E

<400> 3

Ctttctcaaa cacaaactca c 21

<210> 31

<211> 30

<212> RNA

<213> SARS-CoV-2_ORF3a-26144

<400> 1

Acgttggatg tgagcctgaa gaacatgtcc 30

<210> 32

<211> 30

<212> RNA

<213> SARS-CoV-2_ORF3a-26144

<400> 2

Acgttggatg gtagtcgtcg tcggttcatc 30

<210> 33

<211> 22

<212> RNA

<213> SARS-CoV-2_ORF3a-26144

<400> 3

Ccgcttaaca actccggatg aa 22

<210> 34

<211> 31

<212> RNA

<213> SARS-CoV-2_ORF1ab-8782

<400> 1

Acgttggatg gtcattagta taactaccac c 31

<210> 35

<211> 30

<212> RNA

<213> SARS-CoV-2_ORF1ab-8782

<400> 2

Acgttggatg gtcactcgtg acatagcatc 30

<210> 36

<211> 22

<212> RNA

<213> SARS-CoV-2_ORF1ab-8782

<400> 3

Ctgattttga cacatggttt ag22

<210> 37

<211> 30

<212> RNA

<213> SARS-CoV-2_ORF3a-25563

<400> 1

Acgttggatg agagtgctag ttgccatctc 30

<210> 38

<211> 30

<212> RNA

<213> SARS-CoV-2_ORF3a-25563

<400> 2

Acgttggatg ccctttcgga tggcttattg 30

<210> 39

<211> 23

<212> RNA

<213> SARS-CoV-2_ORF3a-25563

<400> 3

Ccccaacttc ttgctgtttt tca 23

<210> 40

<211> 30

<212> RNA

<213> MERS-CoV

<400> 1

Acgttggatg aagtccatgg ctgcaactcg 30

<210> 41

<211> 30

<212> RNA

<213> MERS-CoV

<400> 2

Acgttggatg aagcatgaaa tcccagccac 30

<210> 42

<211> 23

<212> RNA

<213> MERS-CoV

<400> 3

Gtatccaccg tagaactttg tag 23

<210> 43

<211> 30

<212> RNA

<213> SARS-CoV-2_ORF8-28144

<400> 1

Acgttggatg aggctggttc taaatcaccc 30

<210> 44

<211> 30

<212> RNA

<213> SARS-CoV-2_ORF8-28144

<400> 2

Acgttggatg acccaattta ggttcctggc 30

<210> 45

<211> 23

<212> RNA

<213> SARS-CoV-2_ORF8-28144

<400> 3

Aatggcaatt aattgtaaaa ggt 23

<210> 46

<211> 30

<212> RNA

<213> SARS-CoV-2_ORF1ab-11083

<400> 1

Acgttggatg acatcattgc aaaagcagac 30

<210> 47

<211> 30

<212> RNA

<213> SARS-CoV-2_ORF1ab-11083

<400> 2

Acgttggatg gtccagagta ctcaatggtc 30

<210> 48

<211> 24

<212> RNA

<213> SARS-CoV-2_ORF1ab-11083

<400> 3

Ccaatggtct ttgttctttt tttt 24

<210> 49

<211> 30

<212> RNA

<213> SARS-CoV-2_N

<400> 1

Acgttggatg ctacctggcg tggtttgtat 30

<210> 50

<211> 31

<212> RNA

<213> SARS-CoV-2_N

<400> 2

Acgttggatg gaaatgctgg tattgttggt g 31

<210> 51

<211> 24

<212> RNA

<213> SARS-CoV-2_N

<400> 3

Ggggtgtaac tggtatgatt tcgg 24

<210> 52

<211> 30

<212> RNA

<213> SARS-CoV

<400> 1

Acgttggatg tggagaaatg tttacgcagg 30

<210> 53

<211> 29

<212> RNA

<213> SARS-CoV

<400> 2

Acgttggatg caggctctat gagtgtctc 29

<210> 54

<211> 25

<212> RNA

<213> HCoV-NL63

<400> 3

Ggctctatga gtgtctctat agaaa 25

<210> 55

<211> 30

<212> RNA

<213> HCoV-HKU1

<400> 1

Acgttggatg tggcctgtaa tggccataag 30

<210> 56

<211> 29

<212> RNA

<213> HCoV-HKU1

<400> 2

Acgttggatg cataatctgt acgataaac 29

<210> 57

<211> 26

<212> RNA

<213> HCoV-HKU1

<400> 3

Cccctaagcg tttttgtaaa ttgcgt 26

<210> 58

<211> 30

<212> RNA

<213> HCoV-OC43

<400> 1

Acgttggatgt gatacacaag tggataagc 30

<210> 59

<211> 30

<212> RNA

<213> HCoV-OC43

<400> 2

Acgttggatg gttaaagact gatagtgttc 30

<210> 60

<211> 27

<212> RNA

<213> HCoV-OC43

<400> 3

Tggtctttta atagaacgat ttgtaag 27

<210> 61

<211> 30

<212> RNA

<213> SARS-CoV-2_S-23012

<400> 1

Acgttggatg acgttggatg gtgggttgga aaccatatga 30

<210> 62

<211> 42

<212> RNA

<213> SARS-CoV-2_S-23012

<400> 2

Acgttggatg acgttggatg gaaatctatc agaggccggt ag 42

<210> 63

<211> 19

<212> RNA

<213> SARS-CoV-2_S-23012

<400> 3

Gacaccttgt aatggtgtt 19

Claims (10)

1. A kit for detecting 7 human coronaviruses is characterized by comprising primers and probes for detecting SARS-CoV-2 virus molecular typing and variant strains, and the sequences of the primers and probes are shown in SEQ ID NO.1-SEQ ID NO.9, SEQ ID NO.16-SEQ ID NO.24, SEQ ID NO.31-SEQ ID NO.39, SEQ ID NO.43-SEQ ID NO.48 and SEQ ID NO.61-SEQ ID NO. 63.

2. The kit of claim 1, wherein the kit comprises primers and probes for detecting human coronavirus 229E, NL63, HKU1, OC43, SARS-CoV, MERS-CoV, and SARS-CoV-2, wherein SARS-CoV-2 virus comprises S, L, V, G, GH, GR, GV, and GRY typing, and UK, south Africa, and Barceli variants having the sequences of the primer sets and probes shown in SEQ ID No.1-SEQ ID No. 63.

3. The kit of claim 1, further comprising detection means and reagents, such as reagents required for PCR, such as solvents, enzymes, and control substances, as required, wherein the means may comprise, instructions for use, cotton swab, holder, reagent bottle, test paper, and other necessary kit for detection.

4. The kit of claim 2, comprising a reaction buffer mixture, RNA reverse transcriptase and polymerase, shrimp alkaline phosphatase reaction components, and single base extension reaction components.

5. The kit of claim 1, wherein the primers and probes, and the reagents and tools are used in an amount sufficient for testing a sample for 1 person or for testing a sample for multiple persons.

6. The kit of claim 1, wherein the amount of the multi-dose sample is 5, 10, 20, 50, 100, 1000 doses to meet the screening requirement.

7. The special primer for detecting 7 kinds of human coronavirus is selected from the following group: SEQ ID NO.28-SEQ ID NO.30, SEQ ID NO.25-SEQ ID NO.27, SEQ ID NO.55-SEQ ID NO.57, SEQ ID NO.58-SEQ ID NO.60, SEQ ID NO.52-SEQ ID NO.54, SEQ ID NO.40-SEQ ID NO.42, SEQ ID NO.13-SEQ ID NO.15 and SEQ ID NO.49-SEQ ID NO. 51.

8. The special primer for detecting SARS-CoV-2 molecular typing and variant strain detection is selected from: SEQ ID NO.1 to SEQ ID NO.9, SEQ ID NO.16 to SEQ ID NO.24, SEQ ID NO.31 to SEQ ID NO.39, SEQ ID NO.43 to SEQ ID NO.48, SEQ ID NO.61 to SEQ ID NO. 63.

9. The primer set according to claim 5, wherein said molecular typing of SARS-CoV-2 is S, L, V, G, GH, GR, GV, or GRY type, and said SARS-CoV-2 variant is a Brazil variant, a south Africa variant, or a British variant.

10. A method for detecting 7 human coronaviruses, as well as molecular typing and variant detection of SARS-CoV-2 virus, for non-diagnostic disease purposes, comprising the use of the kit of claim 1 of the present invention, said method comprising the steps of:

1) collecting a sample;

2) extracting viral RNA in the sample;

3) performing PCR amplification by using the virus RNA as a template;

4) detecting the PCR amplification product by using a mass spectrometer, and judging the result;

wherein, the sample in the sample collection in the step 1) is a sample to be detected, and the source of the sample comprises a human nasopharynx swab, saliva, a sputum sample, an alveolar lavage fluid sample, a blood sample, lower respiratory secretion, a stool sample or an environment sample;

wherein, the PCR amplification in step 3) requires the use of primers and probes, which can be used for detecting 7 human coronaviruses, and they are: human coronavirus 229E, NL63, HKU1, OC43, SARS-CoV, MERS-CoV and SARS-CoV-2, wherein the SARS-CoV-2 virus comprises S, L, V, G, GH, GR, GV and GRY typing, as well as UK variant, south Africa variant and Brazilian variant;

the sequences of the primer and the probe are shown in SEQ ID NO.1-SEQ ID NO. 63.

Images