CN112029906A

CN112029906A - Two-dimensional code detection method for distinguishing SARS-CoV and SARS-CoV2 virus based on SNP

Info

Publication number: CN112029906A
Application number: CN202010874232.5A
Authority: CN
Inventors: 曹晓梅; 郭军华; 方志强; 张晓龙; 李颖; 慈颖; 刘莹莹
Original assignee: Chinese Academy of Inspection and Quarantine CAIQ
Current assignee: Chinese Academy of Inspection and Quarantine CAIQ
Priority date: 2020-08-27
Filing date: 2020-08-27
Publication date: 2020-12-04
Anticipated expiration: 2040-08-27
Also published as: CN112029906B

Abstract

The present invention relates to virus detecting method, and is especially the detection and identification of SARS-CoV and SARS-CoV2 virus. The invention also relates to a general kit capable of detecting and identifying SARS-associated coronavirus by SNP, which comprises the polynucleotides of SEQ ID NO.1-SEQ ID NO. 36.

Description

Two-dimensional code detection method for distinguishing SARS-CoV and SARS-CoV2 virus based on SNP

The technical field is as follows:

the present invention relates to virus detecting method, and is especially the detection and identification of SARS-CoV and SARS-CoV2 virus.

Background art:

SARS-english abbreviation of severe acute respiratory syndrome. The SARS virus is a variant of coronavirus and is the causative agent of SARS. The variant coronavirus is related to influenza virus, but is a very unique coronavirus, and the coronavirus is the virtuous of severe acute respiratory syndrome (SARS, infectious atypical pneumonia) abused worldwide in winter 2002 to spring 2003. In 2013, an international research team in Shizhenli area, a institute of Wuhan virus of Chinese academy of sciences, China, isolated a SARS-like coronavirus (SARS-like CoV) with high homology to SARS virus, and the sequence similarity of the SARS-like coronavirus and the SARS-like coronavirus is 79%, so that it was further confirmed that Hepialus chinensis is the source of SARS virus. However, there is already epidemiological evidence and bioinformatics analysis that shows that the paguma larvata in the wildlife market is a direct source of SARS coronavirus. In Guangdong area, the paguma larvata in winter of that year carries the virus. This suggests that the paguma larvata may be only an intermediate host for the virus, which may be infected by the horseshoe bats from which the virus was obtained. In summary, the search for the source of SARS virus continues, and there is still a need for a full human co-effort to completely eradicate SARS.

SARS-CoV-an English abbreviation for coronavirus responsible for acute respiratory infectious disease. SARS virus belongs to the genus of coronavirus belonging to the order of nested virus, family of coronaviridae, and is a coronavirus of subgroup B of the genus beta.

SARS-CoV-2-the International Committee for viral Classification on Taxomy of viroses, ICTV, announces that the formal classification of the novel coronavirus (2019-nCoV) is known as Severe acute respiratory syndrome coronavirus2 (SARS-CoV-2, syndrome coronavirus 2). The World Health Organization (WHO) announces on the same day that the official name of the disease caused by this virus is "2019 coronavirus disease (COVID-19)". In addition, nCoV-2019 enters the cell and is the same as SARS-CoV, and is ACE 2.

Laboratory detection techniques for the virus include nucleic acid detection and serological antibody detection. Wherein the nucleic acid detection comprises: a real-time fluorescence RT-PCR method, a constant-temperature amplification chip method, a constant-temperature amplification-real-time fluorescence method, a hybridization capture immunofluorescence method, a combined probe anchoring polymerization sequencing method, an RNA capture probe method, an RNA constant-temperature amplification-gold probe chromatography method and a double-amplification method. Serological antibody detection includes:

a magnetic particle chemiluminescence method for IgM/IgG antibody detection and a colloidal gold method for IgM/IgG antibody detection.

The methods of the present application fall within the scope of nucleic acid detection. The existing kit detects reagents with targets marked by a single target segment (ORF1ab), a double target segment (ORF1ab, N protein) and a triple target segment (RdRp/ORF1ab, N protein and E protein) and is used for detecting SARS-CoV-2. There is a need for a method to synchronously detect SARS-CoV and SARS-CoV2 virus and distinguish them so as to identify SARS virus accurately and quickly in time for use.

The invention selects 1 species-specific SNP conservative sites which can distinguish SARS-CoV and SARS-CoV2 from 10 genes (5 'UTR, L, RdRp, Hel, EC3, S, M, N, Orf10 and 3' UTR) by analyzing the whole genome DNA barcode sequences of three thousand more cases of SARS-CoV2 and three hundred more cases of SARS-CoV related viruses. According to the characteristic that the DNA sequence is composed of 4 basic groups (ATCG), the detection design not only contains the respective specific basic groups of the above two SARS viruses, but also edits the other 2 basic groups which do not appear at present to be used for monitoring novel SARS-CoV-n which possibly appears in the future, thereby establishing the SARS-CoV related virus RNA two-dimensional code identification mark.

The 10 SNP loci are finely sequenced by adopting an ultra-high sensitivity PCR-MS mass spectrum SNP typing technology, and the SARS-CoV related coronavirus can be detected and identified in an ultra-wide dynamic range according to the molecular weight difference of four basic groups (ATCG) extended by a single point of a probe. The virus can be detected at one time from single molecule to billions of molecules. The single sample is simultaneously detected by 10 probes, namely 1 sample is simultaneously subjected to 5-10 times of routine PCR detection, so that the low-load virus missing detection caused by the current commercial reagent 1-2 probe PCR can be effectively avoided. The RNA two-dimensional code identification mark of the 10 SNP loci can be used for detecting and identifying SARS-CoV and SARS-CoV-2, and can be used for monitoring and early warning novel coronavirus SARS-CoV-3, 4, which possibly occurs in the future according to the super-species genetic variation of the 10 SNP loci.

The single sample is simultaneously detected by 10 probes, namely 1 sample is simultaneously subjected to 5-10 times of routine PCR detection once, so that the low-load SARS-CoV-2 virus omission factor caused by the current commercial reagent 1-2 probe PCR detection can be effectively avoided.

The invention content is as follows:

the invention provides a general kit capable of detecting and identifying SARS-related coronavirus through SNP, which not only can accurately detect and distinguish the existing SARS-CoV and SARS-CoV-2 viruses with ultrahigh sensitivity, but also can be used for quickly, effectively and timely monitoring and early warning the novel coronavirus SARS-CoV-3, 4, n which may occur in the future, thereby providing a powerful rapid detection tool for preventing the novel coronavirus and the variant thereof from attacking again in the future for human beings.

The invention screens out species-specific SNP conservative loci capable of distinguishing SARS-CoV and SARS-CoV2 from 10 gene regions (5 'UTR, L, RdRp, Hel, EC3, S, M, N, Orf10 and 3' UTR) by analyzing the whole genome RNA barcode sequences of three thousand more cases of SARS-CoV2 and three hundred more cases of SARS-CoV related viruses, and establishes a SARS-CoV related virus DNA two-dimensional code identification mark.

The invention further designs ten groups of primers and probes, targets 10 SNP sites respectively, and adopts multiple PCR-MS fine sequencing. According to the four-base single point extension combination, the SARS-CoV and SARS-CoV-2 are distinguished, and the possible SARS-CoV-n in the future is predicted.

Table 1 below shows 10 gene regions (5 'UTR, L, RdRp, Hel, EC3, S, M, N, Orf10 and 3' UTR) and their corresponding 10 SNP sites

Table 1. SARS-CoV related virus DNA two-dimensional code recognition table

After the corresponding relation is researched, the invention further provides a detection method, and the detection method adopts Sequenom

The SNP detection process combines multiple PCR technology,

iPLEX single base extension technology, using plasmid DNA carrying SARS-CoV and SARS-CoV-2 nucleic acid as template, amplifying by PCR technology, and then using specific extension primer and PCR product to make single base extension reaction. Because the different tail end bases of the extension products cause the difference of the molecular weight of the extended products due to the different bases of the polymorphic sites, the base difference caused by SNP polymorphism is reflected by the difference of the molecular weight, the size of the molecular weight of the extension products is detected by a mass spectrometry technology, and the SNP typing detection is carried out by judging the difference of the molecular weight by applying special analysis software.

The invention aims to provide a universal kit capable of detecting and identifying SARS-associated coronavirus through SNP, which is characterized in that the kit comprises the polynucleotides of SEQ ID NO.1-SEQ ID NO. 36.

The kit of the invention is used for distinguishing SARS-CoV from SARS-CoV-2 and predicting SARS-CoV-n which may appear in the future. The kit of the invention is applied to the detection and identification of SARS-associated coronavirus.

The detection method comprises the following steps:

step 1, RNA extraction from samples

Extracting RNA from tissue, cell, saliva, extract and blood sample.

The extraction method belongs to the prior art, and uses a commercial extraction kit to extract RNA in tissues, cells or blood samples and stores the RNA at the temperature of minus 80 ℃ for later use.

Step 2, PCR amplification

The primers used in the method are designed by the inventor and comprise the following components:

sequence numbering	Primer name	Sequence (5'to 3')
			SEQ ID NO.1	P2_1	acgttggatg mtcttctgya grctgcttac
SEQ ID NO.2	P2_2	acgttggatg atggttgagc tggtwgcaga
			SEQ ID NO.3	P2_3	acgttggatg gttatgagga tcaagatgca c
SEQ ID NO.4	P2_4	acgttggatg gcrttctgtg aattataagg
			SEQ ID NO.5	P2_5	acgttggatg ccctgttgtc catcaaagtg
SEQ ID NO.6	P2_5_2	acgttggatg ccggcgtgtc catcaaagtg
			SEQ ID NO.7	P2_6	acgttggatg aagrtgrtag ccctttccac
SEQ ID NO.8	P2_7	acgttggatg gtaataagaa agcgttcgtg
			SEQ ID NO.9	P2_8	acgttggatg cggccaatgt ttgtaatcag
SEQ ID NO.10	P2_9	acgttggatg gtctactctt gtgcagaatg
			SEQ ID NO.11	P2_10	acgttggatg tagggaggac ttgaaagagc
SEQ ID NO.12	P2_11	acgttggatg tgcagatttg gacctgcgag
			SEQ ID NO.13	P1_1	acgttggatg ccatcttacc tttcggtcac
SEQ ID NO.14	P1_2	acgttggatg tacgaagaag aacmttgcgg
			SEQ ID NO.15	P1_3	acgttggatg gcgagctcta ttctttgcac
SEQ ID NO.16	P1_4	acgttggatg acrcatgatg tttcatctgc
			SEQ ID NO.17	P1_5	acgttggatg ggrtttacaa acaatttga
SEQ ID NO.18	P1_6	acgttggatg tctgctaatc ttgctgctac
			SEQ ID NO.19	P1_7	acgttggatg taggacgctg tgacatcaag
SEQ ID NO.20	P1_7_2	acgttggatg tagggcgctg tgacattaag
			SEQ ID NO.21	P1_8	acgttggatg tggtccagaa caaacccaag
SEQ ID NO.22	P1_9	acgttggatg gtgagattaa agttaactac
			SEQ ID NO.23	P1_9_2	acgttggatg gtgagattaa agttaactaa
SEQ ID NO.24	P1_10	acgttggatg ctgtacmctc gatcgtactc
			SEQ ID NO.25	P1_11	acgttggatg atagcaacaa ctgaatagcc

And carrying out PCR amplification by taking cDNA after reverse transcription of the sample RNA as a PCR template. The PCR amplification adopts a multiple PCR technology, which is the prior art, for example, a primer mixture prepared by the primers is added into a multiple PCR reaction system, each pair of primers is respectively combined at the corresponding part of a template, and more than one target fragment is finally amplified, for example: adding relevant primers and reagents into the PCR reaction plate, placing the PCR reaction plate on a PCR instrument, and starting a PCR reaction.

Step 3, alkaline phosphatase (SAP) treatment of PCR product

The method is the prior art, and comprises the following steps: after the PCR reaction is finished, the PCR product is treated with SAP to remove free dNTPs in the system. Such as: alkaline phosphatase treatment reaction solution was prepared and added to the PCR reaction plate. The well plate was placed on a compatible PCR instrument, and the PCR instrument was started for alkaline phosphatase treatment by setting the reaction conditions.

Step 4, Single base extension

sequence numbering	Primer name	Sequence (5'to 3')
			SEQ ID NO.26	P3_1_11	gagcgggttc tgac
SEQ ID NO.27	P3_2_1	gcgtccgtgt tgcag
			SEQ ID NO.28	P3_3_10	agccaccaca ttttca
SEQ ID NO.29	P3_4_4	gcttttctcc aagcagg
			SEQ ID NO.30	P3_5_7	gagcgttcgt gatgtagc
SEQ ID NO.31	P3_6_8	cagttccttg tctgattag
			SEQ ID NO.32	P3_7_2	ggcattcagt acggtcgtag
SEQ ID NO.33	P3_8_3	ctagcaagat gcacttttcg c
			SEQ ID NO.34	P3_9_5	aaagccacat tttctaaact ctg
SEQ ID NO.35	P3_10_9	gtgcagaatg aattctcgta acta
			SEQ ID NO.36	P3_11_6	aatccactct ttttgattgt ccaag

The method is the prior art, and comprises the following steps: preparing single base extension reaction liquid, adding the single base extension reaction liquid into the PCR product treated by the SAP, placing the PCR product on a PCR instrument, setting reaction conditions, and starting the PCR instrument to carry out single base extension reaction.

Step 5, resin purification

The method is the prior art, and comprises the following steps: spreading the resin into a resin board; adding water into corresponding holes of the extension product; buckling the resin into an extension product plate, sealing the film, and uniformly mixing the resin and the extension product plate at a low speed for 30 minutes to ensure that the resin is fully contacted with reactants; and (4) centrifuging.

Step 6, chip spotting

The method is the prior art, and comprises the following steps: the MassARRAYANodeispenser RS1000 spotter was started and the resin purified extension product was transferred to a SpectroCHIP chip.

Step 7, mass spectrometric detection

The method is the prior art, and comprises the following steps: the spotted SpectroCHIP was analyzed by MALDI-TOF (matrix-assisted laser desorption ionization time of flight mass spectrometry), and the detection results were analyzed by TYPER 4.0 software (sequenom) and outputted.

The iPLEX is an SNP typing technique, the related technique belongs to the mature prior art, the invention is characterized in that the technique is used for distinguishing SARS-CoV and SARS-CoV-2 and predicting SARS-CoV-n which may appear in the future, therefore, the invention is mainly characterized in that primers and the judgment of experimental results are provided.

The more specific detection method of the invention is as follows:

first step RNA extraction and iPLEX Pre-PCR amplification

The purpose is as follows: the nucleic acid fragment containing the SNP site region was amplified by PCR.

1. Preparation work

(1) Primer dilution and mixing

Except for the probe (iPLEX primer), the dry powder primer is centrifuged at 12000r/min for 5min, the primer is prepared into 100 mu M solution according to the processing information of the primer and nuclease-free water, 1 mu M or 0.5 mu M primer mixture (PrimerMix) is prepared, and 1 mu M PrimerMix is taken as an example to prepare the following:

100 μ l 1 μ M PrimerMix: n primers (100. mu.M) 1. mu.l each

ddH₂O (100-n)μl

1ml 1. mu.M PrimerMix: each 10. mu.l of n primers (100. mu.M)

ddH₂O (1000-n*10)μl

(2) Template preparation

The extracted sample RNA is reverse transcribed by a reverse transcription reagent, and cDNA obtained after reverse transcription is used as a template for PCR amplification.

(3) Sterilized double distilled water was used as a negative control.

2. System preparation (5. mu.l)

After the iPLEX pre-PCR reaction reagent is melted, the mixture is centrifuged after vortex oscillation, and added according to the following sequence:

the mixture was dispensed into 96-or 384-well plates, 3. mu.l/well, and added to the bottom of the tube. Adding 2 μ l sample cDNA template, using water as negative control, sealing membrane, mixing, centrifuging, and placing into PCR instrument for pre-amplification reaction.

3. Reaction procedure

If the next experiment is not carried out on the same day, the product is stored at-20 ℃.

Second step SAP enzyme

The purpose is as follows: dNTPs unused in PCR reactions prior to digestion

1. Preparation work

The SAP reaction reagent and the pre-PCR product were removed, thawed and centrifuged briefly.

2. System preparation

Preparation of SAP reaction MIX, prepared according to the following table:

mu.l of the above mixture (7. mu.l in total volume after adding the mixture) was added to each well of 96-well plate/384-well plate for the pre-PCR reaction, and after the addition, the membrane was sealed, and the mixture was mixed and centrifuged.

3. Reaction conditions

37℃ 40min

85℃ 5min

Storing at 4 deg.C

The samples were stored at-20 ℃ before the next experiment.

Third step iPLEX elongation reaction

A single base extension reaction is performed with the PCR product using a specific extension primer.

1. Preparation work

(1) And taking out the 96-well plate/384-well plate after SAP reaction, and thawing a specific primer (probe) and an iPLEX reaction reagent (iPLEX Buffer Plus, iPLEX Termination mix and iPLEX Pro enzyme) for later use.

(2) Inputting the molecular weight and initial concentration of the specific Primer and the volume of the Primer Mix to be prepared into a Linear Primer Adjustment table, calculating the addition amount of each Primer and water, and preparing the iPLEX extended Primer Mix according to the addition amount.

(3) In the case of newly designed primers and experiments, UEP check is recommended. And (3) subpackaging 20 mu l of the Primer mix to 8-row tubes, carrying out spotting and mass spectrum detection, observing a spectrogram, and judging whether all primers have mass spectrum peaks or not, and judging whether the difference between the highest peak and the lowest peak is larger than 1/2, wherein the peak height of the lowest peak is not smaller than that of the highest peak. If the requirement is not met, the adjustment is needed.

2. System preparation

An iPLEX extension reaction system, prepared according to the following table

Adding 2 mu l of the mixed solution into each 96-hole plate/384-hole plate after SAP reaction, sealing the membrane, uniformly mixing, centrifuging, and carrying out iPLEX reaction.

3. Reaction procedure

Fourth resin purification

1. The reaction plate was aligned to the sample plate and the sample loading position was looked good. And (3) taking 1 spoon of resin on the sample plate by using a long spoon, filling the corresponding hole position of the sample adding hole with the resin, and scraping off the redundant resin by using a scraper. And (4) picking out the resin in the hole position without the sample application by using a gun head.

2. Add 16. mu.l of water to each well of the sample reaction plate, seal the membrane, and centrifuge.

3. Removing the membrane of the sealing plate, slightly putting the sample reaction plate on the sample plate, pinching, slightly inverting, slightly beating the sample plate to make the resin fall into the corresponding hole of the sample plate, and sealing the membrane.

4. Fixing the sample plate on a mixer at room temperature, and rotating and mixing for 15-30 min.

5. The sample plate was removed and centrifuged at 2600r/min for 3 min.

The fifth step of spotting

1. If a 96-well plate is used without a skirt, the plate is placed on a skirted converter. The setup of the spotter has been adjusted to this plate.

2. Volume Check was performed with a spotting machine to find a suitable dispensing speed. Suitable volumes for spotting are 8-12 nL. + -. 25%.

3. Volume check with authentic samples on plate, then MassARRAY^TMThe RS1000 spotter spots the sample onto a 96-spot SpectroCHIP.

Sixth Mass Spectrometry Massarray

The operation is carried out according to the simple operation guide of the Massarray system.

From the results obtained from the mass spectrometer, the method for discriminating SARS-CoV from SARS-CoV-2 and predicting SARS-CoV-n which may appear in the future was determined as follows:

if more than 2 sites report the same virus type, the corresponding virus type can be judged.

The software reports directly that the result of detection is SARS-CoV or SARS-CoV-2, and SARS-CoV-n, based on the result of detection of the SNP base and according to the two-dimensional code recognition rule assigned by the editor (see Table 1). When SARS-CoV-n is present, immediate sequencing is recommended.

The following explains the terms of the terms used in the detection method of the present invention:

SARS-CoV associated Virus Whole genome RNA Bar code sequence-a standard, sufficiently mutated, easily amplified and relatively short gene fragment that can represent this species within a coronavirus causing acute respiratory infectious disease.

10 Gene regions (5 'UTR, L, RdRp, Hel, EC3, S, M, N, Orf-10 and 3' UTR)

5' UTR-the 5' non-coding Region (5' Untranstrained Region) is an Untranslated RNA segment of a hundred nucleotides preceding the translation initiation codon

L-leader protein (leader protein)

RdRp-RNA dependent RNA polymerase gene

Hel-helicase (helicase)

EC 3-endoribonuclease (endoRNAse) specifically attacks the bond between the phosphate at C '-3 of a pyrimidine nucleotide and the C' -5 of an adjacent nucleotide in RNA

S-Can encode to produce the structural protein: surface glycoprotein (surface glycoprotein), i.e., spike protein

M-encoding producing structural proteins: membrane protein (membrane glycoprotein)

N-Can encode to produce the structural protein: nucleocapsid phosphoprotein (nucleocapsid phosphoprotein)

Orf 10-encoding accessory protein 10(ORF10 protein)

3 'UTR-3' end non-coding region sequence

SNP-Single nucleotide polymorphism site

iPLEX pre-PCR reaction reagent:

H₂o HPLC grade: water, HPLC grade (high performance liquid chromatography grade reagent).

10 × PCR Buffer: 10 XPCR reaction buffer containing Tris-HCl, MgCl₂And the like.

dNTP mix: is a mixed solution of dATP, dCTP, dGTP and dTTP and is used for PCR amplification reaction.

Primer mix: and (3) mixing the multiple PCR primers.

hot star Taq polymerase: hot start Taq polymerase.

SAP reaction agent:

H2O (autoclaved): water, which is sterilized by high-pressure steam.

10 × SAP Buffer: SAP reaction buffer containing Bis-Tris HCl, MgCl₂，ZnCl₂。

SAP enzyme: shrimp alkaline phosphatase, degradation of PCR reaction dNTP.

iPLEX reaction reagent:

iPLEX Buffer Plus: the iPLEX extends the reaction buffer solution, providing/maintaining the reaction environment.

iPLEX Termination mix: the iPLEX reaction terminated the mixed solution.

iPLEX extended Primer mix: iPLEX extends the reaction primer mixture.

iPLEX Pro enzyme: iPLEX elongation reaction enzyme.

The apparatus used was: MassARRAY^TMRS1000 spotting instrument: and the sample applicator transfers the sample to the corresponding position of the chip.

Massarray system: a bench-top MALDI-TOF mass spectrometer, a data processing and analysis software package.

Reference sequence:

NC-004718 GenBank accession number of SARS-CoV reference sequence

NC-045512 SARS-CoV-2 reference sequence GenBank accession number

The invention provides a detection kit, which can select one SNP from 10 genomes as a target respectively and detect the corresponding relation of the SNP, wherein a core detection reagent comprises:

the PCR primers are shown in the following table:

primer name	Sequence (5'to 3')
		P2_1	acgttggatg mtcttctgya grctgcttac
P2_2	acgttggatg atggttgagc tggtwgcaga
		P2_3	acgttggatg gttatgagga tcaagatgca c
P2_4	acgttggatg gcrttctgtg aattataagg
		P2_5	acgttggatg ccctgttgtc catcaaagtg
P2_5_2	acgttggatg ccggcgtgtc catcaaagtg
		P2_6	acgttggatg aagrtgrtag ccctttccac
P2_7	acgttggatg gtaataagaa agcgttcgtg
		P2_8	acgttggatg cggccaatgt ttgtaatcag
P2_9	acgttggatg gtctactctt gtgcagaatg
		P2_10	acgttggatg tagggaggac ttgaaagagc
P2_11	acgttggatg tgcagatttg gacctgcgag
		P1_1	acgttggatg ccatcttacc tttcggtcac
P1_2	acgttggatg tacgaagaag aacmttgcgg
		P1_3	acgttggatg gcgagctcta ttctttgcac
P1_4	acgttggatg acrcatgatg tttcatctgc
		P1_5	acgttggatg ggrtttacaa acaatttga
P1_6	acgttggatg tctgctaatc ttgctgctac
		P1_7	acgttggatg taggacgctg tgacatcaag
P1_7_2	acgttggatg tagggcgctg tgacattaag
		P1_8	acgttggatg tggtccagaa caaacccaag
P1_9	acgttggatg gtgagattaa agttaactac
		P1_9_2	acgttggatg gtgagattaa agttaactaa
P1_10	acgttggatg ctgtacmctc gatcgtactc
		P1_11	acgttggatg atagcaacaa ctgaatagcc

Single base extension probes are as follows:

primer name	Sequence (5'to 3')
		P3_1_11	gagcgggttc tgac
P3_2_1	gcgtccgtgt tgcag
		P3_3_10	agccaccaca ttttca
P3_4_4	gcttttctcc aagcagg
		P3_5_7	gagcgttcgt gatgtagc
P3_6_8	cagttccttg tctgattag
		P3_7_2	ggcattcagt acggtcgtag
P3_8_3	ctagcaagat gcacttttcg c
		P3_9_5	aaagccacat tttctaaact ctg
P3_10_9	gtgcagaatg aattctcgta acta
		P3_11_6	aatccactct ttttgattgt ccaag

And optionally (different brands of agents) the following components: buffer, eluent, preservative, enzyme, etc., such as the following:

primer Mix for iPLEX pre-PCR reaction

10 XPCR Buffer (10 XPCR reaction Buffer)

Hot star Taq polymerase (Hot Start Taq polymerase)

10 XSAP Buffer (10 XSAP shrimp alkaline phosphatase Buffer)

SAP enzyme (SAP enzyme, shrimp alkaline phosphatase)

iPLEX Buffer Plus (iPLEX single base extension reaction Buffer)

iPLEX Termination mix (iPLEX single base extension Termination mix)

iPLEX extended Primer mix (iPLEX single base extension Primer mixture)

iPLEX Pro enzyme (iPLEX high fidelity enzyme)

Preferred are the following components:

10 XPCR Buffer (containing 20mM MgCl)₂) (10 XPCR reaction buffer, containing 20mM MgCl)₂)

25mM MgCl₂(25mM magnesium chloride)

25mM dNTP mix (25mM dNTP mix)

1 μ M Primer mix (1 μ M PCR Primer mix)

5U/. mu.l hot star Taq polymerase (5U/. mu.l hot start Taq polymerase)

10 × SAP Buffer (10 × SAP reaction Buffer)

1.7U/. mu.l SAP enzyme (1.7U/. mu.l SAP enzyme, shrimp alkaline phosphatase)

10 × iPLEX Buffer Plus (iPLEX single-base extension reaction Buffer)

5 μ M iPLEX Termination mix (5 μ M iPLEX single base extension Termination mix)

33U/. mu.l iPLEX extended Primer mix (33U/. mu.l iPLEX single base extension Primer mix)

iPLEX Pro enzyme (iPLEX high fidelity enzyme)

The detection method of the invention is superior to the sensitivity of 1-4 target gene detection adopted by all commercial kits at present.

The detection method adopts a genotyping standard 'multiplex PCR-MS flight time mass spectrometer' for developing human 'noninvasive prenatal diagnosis' and 'tumor liquid biopsy'. The instrument has an ultra-wide dynamic range, and can detect single molecules to hundreds of millions of molecules simultaneously. The sensitivity reaches 3-10 copies/reaction, the dynamic range is more than or equal to 9 orders of magnitude, and the low-load virus omission detection by the conventional method can be effectively avoided. Because the fluorescent marker is not needed, the cost for increasing the detection repetition is very low, and the method is suitable for second-line reinspection of the current large-scale rehabilitation patients. Has great significance for monitoring novel SARS-CoV-n in the future.

For the English abbreviations and the names appearing in the specification, the further explanation is as follows:

HPLC Grade: high performance liquid chromatography;

buffer, Buffer solution for providing/maintaining reaction environment;

dNTP mix is a mixed solution of dATP, dCTP, dGTP and dTTP and is used for PCR amplification reaction;

hot star Taq polymerase, hot start Taq polymerase, for PCR amplification;

autoclaved, autoclaved;

enzyme, enzyme;

SAP: shrimp alkaline phosphatase;

termination mix: terminating the reaction mixture;

extended Primer: and (3) extending the primer.

Description of the drawings:

FIG. 1 shows the negative mass control spectrum of heavy PCR (10SARS gene +1RNA mass control);

FIG. 2 shows a mass spectrum of simulated SAR-CoV-2 by the double PCR detection;

FIG. 3 shows a mass spectrum of a simulated SAR-CoV by the double PCR detection;

FIG. 4 shows the result of testing single-gene specimen simulating SARS-CoV-2 and SARS-CoV 5UTR, wherein a: the peak of the negative control appeared at 4600 Da; b: the peak of SARS-COV-2 appears at 4847.2 Da; c: the peak value of SRAS-CoV appears at 4927.1 Da;

FIG. 5 shows the result of testing a single-gene specimen simulating SARS-CoV-2 and SARS-CoV ORF1ab, wherein a: the peak value of the negative control appears at 6173 Da; b: the peak of SARS-COV-2 appears at 6500.1 Da; c: the peak value of SRAS-CoV appears at 6420.2 Da;

FIG. 6 shows the result of testing single-gene preparations simulating SARS-CoV-2 and SARS-CoV RdRp, wherein a: the peak value of the negative control appears at 6381.1 Da; b: the peak of SARS-COV-2 appears at 6652.4 Da; c: the peak value of SRAS-CoV appears at 6668.4 Da;

FIG. 7 shows the results of testing a model SARS-CoV-2 and SARS-CoV ZD single-gene sample, wherein a: the peak value of the negative control appears at 5161.4 Da; b: the peak of SARS-COV-2 appears at 5448.61 Da; c: the peak value of SRAS-CoV appears at 5432.6 Da;

FIG. 8 shows the result of testing a single-gene specimen simulating SARS-CoV-2 and SARS-CoV Exn, wherein a: the peak of the negative control appears at 6966.6 Da; b: the peak of SARS-COV-2 appears at 7237.8 Da; c: the peak value of SRAS-CoV appears at 7293.7 Da;

FIG. 9 shows the result of testing a single-gene specimen simulating SARS-CoV-2 and SARS-CoV S, wherein a: the peak value of the negative control appears at 7581.9 Da; b: the peak of SARS-COV-2 appears at 7909 Da; c: the peak value of SRAS-CoV appears at 7853.2 Da; FIG. 10 shows the results of testing single-gene preparations simulating SARS-CoV-2 and SARS-CoV M, wherein a: the peak value of the negative control appears at 5570 Da; b: the peak of SARS-COV-2 appears at 5841.8 Da; c: the peak value of SRAS-CoV appears at 5817.8 Da; FIG. 11 shows the results of testing single-gene preparations simulating SARS-CoV-2 and SARS-CoV N, wherein a: the peak value of the negative control appears at 5784.8 Da; b: the peak of SARS-COV-2 appears at 6111.9 Da; c: the SRAS-CoV peak appears at 6072 Da; FIG. 12 shows the results of testing a single-gene specimen simulating SARS-CoV-2 and SARS-CoV ORF10, wherein a: the peak value of the negative control appears at 7375.8 Da; b: the peak of SARS-COV-2 appears at 7623 Da; c: the SRAS-CoV peak appears at 7647 Da; FIG. 13 shows the result of testing a single-gene specimen simulating SARS-CoV-2 and SARS-CoV 3UTR, wherein a: the peak value of the negative control appears at 4785.10 Da; b: the peak of SARS-COV-2 appears at 5032.3 Da; c: the SRAS-CoV peak occurs at 5112.2 Da.

The specific implementation mode is as follows:

the invention is further illustrated by the following examples, which are not to be construed as limiting the invention thereto.

Example 1

Example 2

First, preparation of experiment

1. Primer design

Searching and collecting related sequences, performing cluster analysis by using Sequencher5.3 software, selecting appropriate specific Single Nucleotide Polymorphism (SNP) as a target, establishing a typing map, and performing clustering analysis by using the SNP as a target

MassARRAY Assay design 4.0 software, designing multiple PCR specific primers and probes, and synthesizing.

2. A plasmid containing a nucleic acid fragment of the SNP site was synthesized as a mock sample.

3. Reagents, materials and apparatus

Anhydrous ethanol

Nuclease-free water (for PCR amplification and primer dilution)

Pure water (for distilled water, 50% ethanol, resin washing)

Desicator (chip capable of storage)

Mixer (for mixing resin and amplification product in 96-well plate)

Centrifuge (for 96-hole plate/384-hole plate centrifugation)

PCR instrument (ABI9700, ABI2720 can be used for iPLEX PCR amplification, and gradient PCR instrument can be used for single base extension)

96 pore plate/384 pore plate (matching with chip)

Second, the experimental procedure

First step iPLEX Pre-PCR amplification

The purpose is as follows: the DNA containing the SNP site region was amplified by PCR.

1. Preparation work

(1) Primer dilution and mixing

Except for the probe (iPLEX primer), the dry powder primer is centrifuged at 12000r/min for 5min, the primer is prepared into 100 mu M solution according to the processing information of the primer and nuclease-free water, and the solution is mixed to prepare 1 mu M/0.5 mu M primer mixed solution (PrimerMix) which is prepared as follows:

100 μ l PrimerMix: n primers (100. mu.M) 1. mu.l each

ddH₂O (100-n)μl

1ml PrimerMix: each 10. mu.l of n primers (100. mu.M)

ddH₂O (1000-n*10)μl

(2) Template dilution

The suggested template for PCR amplification is 3000-

(3) Sterilized double distilled water was used as a negative control.

2. System preparation (5. mu.l)

After the pre-PCR reaction reagent is melted, the mixture is centrifuged after vortex oscillation, and added according to the following sequence:

the mixture was dispensed into 96-or 384-well plates, 3. mu.l/well, and added to the bottom of the tube. Adding 2 μ l of sample, using water as negative control, sealing membrane, mixing, centrifuging, and placing into PCR instrument for pre-amplification reaction.

Note that: the sample layout position is 6 holes/pins when the samples are printed, and the samples are preferably arranged in a mode of arranging n units with 6 grids as one unit as shown in the figure

3. Reaction procedure

Second step SAP enzyme

The purpose is as follows: dNTPs unused in PCR reactions prior to digestion

1. Preparation work

2. System preparation

Preparation of SAP reaction MIX, prepared according to the following table:

3. Reaction conditions

37℃ 40min

85℃ 5min

Storing at 4 deg.C

The samples were stored at-20 ℃ before the next experiment.

Third step iPLEX elongation reaction

1. Preparation work

(2) Inputting the molecular weight, initial concentration and volume of Primermix to be prepared of the specific primers into a LinearPrimerAdjustment table, calculating the addition amount of each Primer and water, and preparing the iPLEX extended Primer Mix according to the addition amount.

(3) In the case of newly designed primers and experiments, EPU check is recommended. And (3) subpackaging 20 mu l of the Primer mix to 8-row tubes, spotting and hitting mass spectra, observing the spectrograms, and judging whether all primers have mass spectrum peaks or not, and judging whether the difference between the highest peak and the lowest peak is larger than 1/2, wherein the peak height of the lowest peak is not lower than that of the highest peak. If the requirement is not met, the adjustment is needed.

2. System preparation

An iPLEX extension reaction system, prepared according to the following table

3. Reaction procedure

A gradient PCR instrument was used in the experiment, programmed as follows:

fourth resin purification

1. The reaction plate was aligned to the sample plate and the sample loading position was looked good. Placing the sample plate on a piece of A4 paper (smooth surface), taking 1 spoon of resin from the sample plate with a spoon to fill the hole corresponding to the sample hole with resin, and scraping the excess resin with a scraper down to A4 paper for recovery. And (4) picking out the resin in the hole position without the sample application by using a gun head, and recycling the resin together.

3. Removing the sealing film, slightly putting the sample plate on the sample plate, pinching, slightly inverting, slightly beating the sample plate to make the resin fall into the corresponding hole of the sample plate, and sealing the film.

5. The sample plate was removed and centrifuged at 2600r/min for 3min (4000 r/min, 5min in the operating guidelines).

The fifth step of spotting

1. Preparation work

The needle was soaked with absolute ethanol overnight 1 day before the experiment.

2. Spotting process

(1) Adding alcohol: and opening the hatch door, taking out the reagent bottle, adding 50% alcohol, and mounting the reagent bottle by pushing the thumb against the bottom. Note: absolute ethyl alcohol is added into a needle soaking reagent bottle, and 50% ethyl alcohol is added during experiments.

(2) Chip and sample plate (tube) were placed. Note that: the A1 position of the chip and sample plate is located in the lower left corner.

(3) Mapping (design spotting):

and entering a design interface. Clicking create to establish a file, selecting corresponding parameters, and setting a 96-well plate in the experiment as follows:

MTP plate: a sample plate; spectrochi pFormat: chip format; PinFormat: the format of the pin. Clicking 'application' after selection, selecting a sample application position, then performing dot storage, and storing a sample application scheme.

(4) Method (spotting protocol)

And entering a scheme interface.

Setup settings, as follows:

selecting Mapping files, namely files saved in the last step.

SpectroCHIPs: the location where the chip is placed.

Targetvolume (nl): sample application volume was set at 12 nl.

VolumeCheck: setting an upper alarm limit and a lower alarm limit of 5-18nl, and selecting a check volume Enable.

Clean, wash parameter settings as follows:

aspirate/dispense, sample/spotting parameter settings, as follows:

note that: the distance between two printing stations is generally 80-140mm/sec (usually set as 100mm/sec), and needs to be adjusted according to the actual printing volume in the experimental process, and the printing speed is faster and the printing amount is larger. The sample application time (release) can not meet the requirement after the sample application speed is adjusted, and then the sample application time is adjusted. After spotting, the actual spot volume can be viewed, preferably not exceeding 20 nl.

After the setting is finished, clicking back, popping up a window, and clicking application.

(5)Transfer

And entering the rotating plate interface. Step spotting, namely spotting the sample in one step, and after the first needle is spotted, adjusting and then spotting the next needle; if run is selected, all spotting is done automatically according to the previous setup.

Sixth Mass Spectrometry Massarray

Thus, the relationship between the 10 gene regions (5UTR, Orf1a, RdRp, ZD, S protein, 3a, M protein, N protein, Orf-10 and 3UTR) in the following table and the corresponding 10 SNP sites and virus species was found

The results are shown in FIGS. 1-13, and the single-gene mock sample and 11-fold mock sample were detected at each site, and SARS-CoV-2 could be distinguished.

The conclusion is as follows:

the method can be used for the detection and identification of SARS-CoV and SARS-CoV2 virus, and can be used for the prediction of SARS-CoV-n (unknown SARS).

The design of the kit of the invention selects one SNP from 10 genomes as a target, and the detection effect is obviously superior to the sensitivity of 1-4 target gene detection adopted by all commercial kits at present. The detection method adopts a genotyping golden standard 'multiple PCRMS time-of-flight mass spectrometer' for developing human 'noninvasive prenatal diagnosis' and 'tumor liquid biopsy'. The instrument has an ultra-wide dynamic range, and can detect single molecules to hundreds of millions of molecules simultaneously. The sensitivity reaches 3-10 copies/reaction, the dynamic range is more than or equal to 9 orders of magnitude, and the low-load virus omission of the conventional method can be effectively avoided. Because the fluorescent marker is not needed, the cost for increasing the detection repetition is very low, and the method is suitable for the second-line reinspection of the current large-scale rehabilitation patients. Has great significance for monitoring novel SARS-CoV-n in the future.

Claims

1. A universal kit capable of detecting and identifying SARS-associated coronavirus by SNP, comprising the polynucleotide of SEQ ID No.1-SEQ ID No. 36.

2. The kit of claim 1, for differentiating SARS-CoV from SARS-CoV-2 and predicting the likely future appearance of SARS-CoV-n.

3. Use of the kit of claim 1 for the detection and identification of SARS-associated coronavirus.

4. Use according to claim 3, comprising the steps of:

step 1, RNA extraction from samples

Extracting RNA from tissue, cell, saliva, extractive solution, or blood sample,

step 2, PCR amplification

The primers are as follows:

carrying out PCR amplification by taking cDNA reverse transcribed from sample RNA as a PCR template, wherein the PCR amplification adopts a multiple PCR technology, a primer mixture prepared by the primers is added into a multiple PCR reaction system, each pair of primers is respectively combined at the corresponding part of the template, and at least one target fragment is finally amplified,

step 3, alkaline phosphatase (SAP) treatment of PCR product

After the PCR reaction is finished, the PCR product is treated by SAP to remove free dNTPs in the system,

step 4, Single base extension

The primers used were as follows:

Preparing single base extension reaction liquid, adding the single base extension reaction liquid into a PCR product treated by SAP, placing the PCR product on a PCR instrument, setting reaction conditions, starting the PCR instrument to carry out single base extension reaction,

step 5, resin purification

Spreading the resin into a resin board; adding water into corresponding holes of the extension product; buckling the resin into an extension product plate, sealing the film, and uniformly mixing the resin and the extension product plate at a low speed for 30 minutes to ensure that the resin is fully contacted with reactants; the mixture is centrifuged and then is processed by centrifugal separation,

step 6, chip spotting

Starting up

A Nanodiproperser RS1000 sample applicator, the extension product after resin purification is transferred to a SpectroCHIP chip,

step 7, mass spectrometric detection

And analyzing the spotted SpectroCHIP chip by using MALDI-TOF, analyzing the detection result by using TYPER 4.0 software, and outputting the result.

5. Use according to claim 3, comprising the steps of:

first step RNA extraction and iPLEX Pre-PCR amplification

1. Preparation work

(1) Primer dilution and mixing

100 μ l 1 μ M PrimerMix: n primers (100. mu.M) 1. mu.l each

ddH₂O (100-n)μl

1ml 1. mu.M PrimerMix: each 10. mu.l of n primers (100. mu.M)

ddH₂O (1000-n*10)μl

(2) Template preparation

The extracted sample RNA is reverse transcribed by a reverse transcription reagent, cDNA obtained after reverse transcription is used as a template for PCR amplification,

(3) sterilized double distilled water was used as a negative control

2. System preparation (5. mu.l)

subpackaging the above mixed solution into 96-well plate/384-well plate with each well being 3 μ l, adding to the bottom of the tube, adding 2 μ l sample cDNA template, using water as negative control, sealing the membrane, mixing, centrifuging, placing into PCR instrument for pre-amplification reaction,

3. reaction procedure

Second step SAP enzyme

The purpose is as follows: dNTPs unused in PCR reactions prior to digestion

1. Preparation work

Taking out and melting the SAP reaction reagent and the pre-PCR product, centrifuging for a short time,

2. system preparation

Preparation of SAP reaction MIX, prepared according to the following table:

adding 2 mul of the mixed solution into each well of a 96-well plate/384-well plate of the pre-PCR reaction, sealing a membrane after the addition, uniformly mixing and centrifuging,

3. reaction conditions

37℃ 40min

85℃ 5min

Storing at 4 deg.C

The sample is preserved at-20 ℃ before the next experiment,

third step iPLEX elongation reaction

1. Preparation work

(1) Taking out the 96-well plate/384-well plate after SAP reaction, melting the specific primer (probe) and the iPLEX reaction reagent for later use,

(2) inputting the molecular weight and initial concentration of specific primers and the volume of the Primer Mix to be prepared into a Linear Primer Adjustment table, calculating the addition amount of each Primer and water, preparing the iPLEX extended Primer Mix according to the addition amount,

(3) subpackaging 20 μ l of the above Primer mix to 8-row tubes, spotting, detecting by mass spectrometry, observing spectrogram, detecting whether all primers have mass spectrum peak, and difference between the highest peak and the lowest peak, requiring that the peak height of the lowest peak is not lower than 1/2 of the highest peak,

2. system preparation

An iPLEX extension reaction system, prepared according to the following table

Adding 2 μ l of the above mixed solution into each well of 96-384-well plate after SAP reaction, sealing, mixing, centrifuging, carrying out iPLEX reaction,

3. reaction procedure

Fourth resin purification

1. Comparing the reaction plate with the sample plate, observing the sample loading position, loading 1 scoop of resin on the sample plate with a long scoop to fill the hole position corresponding to the sample loading hole with the resin, scraping off the excess resin with a scraper, picking out the resin in the hole position which is not loaded with the sample with a gun head,

2. adding 16 mul of water into each hole of the sample reaction plate, sealing the membrane, centrifuging,

3. removing the sealing film, slightly placing the sample reaction plate on the sample plate, pinching, slightly inverting, lightly tapping the sample plate to make the resin fall into the corresponding hole of the sample plate, sealing the film,

4. fixing the sample plate on a mixer at room temperature, mixing for 15-30min by rotation,

5. taking off the sample plate, centrifuging at 2600r/min for 3min,

the fifth step of spotting

1. If a 96-well plate is used without a skirt, the plate is placed on a skirted converter, the placement of the spotting apparatus is adjusted to the plate,

2. performing volume test with a spotting instrument to find out proper spotting speed, wherein the proper spotting volume is 8-12nL + -25%,

3. volume check with authentic samples on plate, then MassARRAY^TMThe RS1000 spotter spots the sample onto a 96-spot SpectroCHIP chip,

sixth Mass Spectrometry Massarray

The operation is carried out according to Massarray system operation guidelines.

6. The application of claim 3, further comprising a judgment process:

from the results obtained by the mass spectrometer, if more than 2 sites report the same virus type, the corresponding virus type can be judged,

7. use according to claim 3, comprising the steps of:

first step iPLEX Pre-PCR amplification

1. Preparation work

(1) Primer dilution and mixing

100 μ l PrimerMix: n primers (100. mu.M) 1. mu.l each

ddH₂O (100-n)μl

1ml PrimerMix: each 10. mu.l of n primers (100. mu.M)

ddH₂O (1000-n*10)μl

(2) Template dilution

The suggested template for PCR amplification is 3000-

(3) Sterilized double distilled water was used as a negative control.

2. System preparation (5. mu.l)

the mixture was dispensed into 96-or 384-well plates, 3. mu.l/well, and added to the bottom of the tube. Adding 2 μ l of sample, using water as negative control, sealing membrane, mixing, centrifuging, placing into PCR instrument for pre-amplification reaction,

3. reaction procedure

Second step SAP enzyme

1. Preparation work

2. System preparation

Preparation of SAP reaction MIX, prepared according to the following table:

mu.l of the above mixture was added to each well of a 96-well plate/384-well plate for the pre-PCR reaction, and after the addition, the mixture was sealed and mixed well and centrifuged.

3. Reaction conditions

37℃ 40min

85℃ 5min

Storing at 4 deg.C

The samples were stored at-20 ℃ before the next experiment.

Third step iPLEX elongation reaction

1. Preparation work

(1) And taking out the 96-well plate/384-well plate after the SAP reaction, and melting the specific primer (probe) and the iPLEX reaction reagent for later use.

(3) And (3) subpackaging 20 mu l of the Primer mix to 8-row tubes, spotting and hitting mass spectra, observing the spectrograms, and judging whether all primers have mass spectrum peaks, and judging whether the difference between the highest peak and the lowest peak is higher than 1/2 of the highest peak. If the requirement is not met, the adjustment is needed.

2. System preparation

An iPLEX extension reaction system, prepared according to the following table

3. Reaction procedure

A gradient PCR instrument was used in the experiment, programmed as follows:

fourth resin purification

1. The reaction plate was aligned to the sample plate and the sample loading position was looked good. Placing the sample plate on a piece of A4 paper, taking 1 spoon of resin from the sample plate with a long spoon to fill the hole corresponding to the sample hole with resin, and scraping the excess resin with a scraper down to the A4 paper for recycling. And (4) picking out the resin in the hole position without the sample application by using a gun head, and recycling the resin together.

5. The sample plate was removed and centrifuged at 2600r/min for 3 min.

The fifth step of spotting

1. Preparation work

2. Spotting process

(1) Adding alcohol: and opening the hatch door, taking out the reagent bottle, adding 50% alcohol, and mounting the reagent bottle by pushing the thumb against the bottom.

Note: absolute ethyl alcohol is added into a needle soaking reagent bottle, and 50% ethyl alcohol is added during experiments.

(2) The chip and sample plate are placed. Note that: the A1 position of the chip and sample plate is located in the lower left corner.

(3) Mapping (design spotting):

(4) Method (spotting protocol)

And entering a scheme interface.

Setup settings, as follows:

selecting Mapping files, namely files saved in the last step.

SpectroCHIPs: the location where the chip is placed.

Targetvolume: sample application volume was set at 12 nl.

Clean, wash parameter settings as follows:

aspirate/dispense, sample/spotting parameter settings, as follows:

note that: dispenseting, the dispensespeed is generally 80-140mm/sec, and needs to be adjusted according to the actual sample application volume in the experimental process, and the higher the sample application speed is, the more the sample application amount is. The sample application time can not meet the requirement after the sample application speed is adjusted, and then the sample application time is adjusted. After spotting, the actual spot volume can be viewed, preferably not exceeding 20 nl.

(5)Transfer

Sixth Mass Spectrometry Massarray

Operating according to the simple operating guide of the Massarray system,

from the results obtained with the mass spectrometer, the relationship between 10 gene regions and their corresponding 10 SNP sites and virus species was obtained: