CN112029906B - Two-dimensional code detection method for distinguishing SARS-CoV and SARS-CoV2 virus based on SNP - Google Patents
Two-dimensional code detection method for distinguishing SARS-CoV and SARS-CoV2 virus based on SNP Download PDFInfo
- Publication number
- CN112029906B CN112029906B CN202010874232.5A CN202010874232A CN112029906B CN 112029906 B CN112029906 B CN 112029906B CN 202010874232 A CN202010874232 A CN 202010874232A CN 112029906 B CN112029906 B CN 112029906B
- Authority
- CN
- China
- Prior art keywords
- sars
- cov
- iplex
- reaction
- seq
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/70—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage
- C12Q1/701—Specific hybridization probes
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6844—Nucleic acid amplification reactions
- C12Q1/6858—Allele-specific amplification
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/156—Polymorphic or mutational markers
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/16—Primer sets for multiplex assays
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Landscapes
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Immunology (AREA)
- General Engineering & Computer Science (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Microbiology (AREA)
- Molecular Biology (AREA)
- Analytical Chemistry (AREA)
- Physics & Mathematics (AREA)
- Biophysics (AREA)
- Genetics & Genomics (AREA)
- General Health & Medical Sciences (AREA)
- Biotechnology (AREA)
- Biochemistry (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Virology (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
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
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 monitor and early warn novel coronavirus SARS-CoV-3, 4 and … n which may occur in the future according to the super-species genetic variation of the SARS-CoV and the SARS-CoV-2.
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 bases of the polymorphic sites lead to the difference of the molecular weights of the extended products due to the different end bases of the extended products, the base difference caused by SNP polymorphism is reflected by the difference of the molecular weights, and the molecular weight of the extended products is detected by the mass spectrometry technologySize, SNP typing detection was carried out by determining the difference in molecular weight using a dedicated 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:
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.
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.
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.
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.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.
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.
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.
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
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 MassARRAYTMThe 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: MassARRAYTMRS1000 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
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.
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
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, 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
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
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.
2. Add 16. mu.l of water to each well of the sample reaction plate, seal the membrane, and centrifuge.
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.
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 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
The operation is carried out according to the simple operation guide of the Massarray system.
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 (3)
1. A kit capable of differentiating SARS-CoV from SARS-CoV-2 virus by SNP, which comprises the polynucleotide of SEQ ID No.1 to SEQ ID No. 36.
2. Use of the polynucleotide of SEQ ID No.1-SEQ ID No.36 of claim 1 for the preparation of a kit for differentiating SARS-CoV from SARS-CoV-2 virus.
3. The kit of claim 1, comprising the following components:
10 XPCR Buffer containing 20mM MgCl2
25mM MgCl2
25mM dNTP mix
1µM Primer mix
5U/µl hot star Taq polymerase
10×SAP Buffer
1.7U/µl SAP enzyme
10×iPLEX Buffer Plus
5µM iPLEX Termination mix
33U/µl iPLEX Extend Primer mix
iPLEX Pro enzyme。
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010874232.5A CN112029906B (en) | 2020-08-27 | 2020-08-27 | Two-dimensional code detection method for distinguishing SARS-CoV and SARS-CoV2 virus based on SNP |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010874232.5A CN112029906B (en) | 2020-08-27 | 2020-08-27 | Two-dimensional code detection method for distinguishing SARS-CoV and SARS-CoV2 virus based on SNP |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN112029906A CN112029906A (en) | 2020-12-04 |
| CN112029906B true CN112029906B (en) | 2021-06-11 |
Family
ID=73580092
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202010874232.5A Active CN112029906B (en) | 2020-08-27 | 2020-08-27 | Two-dimensional code detection method for distinguishing SARS-CoV and SARS-CoV2 virus based on SNP |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN112029906B (en) |
Families Citing this family (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113215231B (en) * | 2021-03-30 | 2022-08-05 | 成都里来生物科技有限公司 | Dual PCR detection method for SARS-CoV and COVID-19 virus |
| WO2023002505A1 (en) * | 2021-07-19 | 2023-01-26 | Indian Council Of Medical Research | Assay for detection of epidemiologically important sars- cov-2 variants |
| CN113846189B (en) * | 2021-11-03 | 2023-05-26 | 安徽医科大学第二附属医院 | Novel coronavirus genotype reference sequence based on GISAID typing, design primer, kit and application |
Citations (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105238853A (en) * | 2015-08-20 | 2016-01-13 | 复旦大学附属华山医院 | Method for simultaneously measuring gene polymorphism of tacrolimus action targets of person |
| CN111139317A (en) * | 2020-03-13 | 2020-05-12 | 欧陆分析技术服务(苏州)有限公司 | Multiplex fluorescent quantitative PCR detection kit and detection method for SARS-COV-2 virus |
| CN111235316A (en) * | 2020-03-23 | 2020-06-05 | 中国检验检疫科学研究院 | Primer probe for identifying novel coronavirus and application of primer probe in triple fluorescence RPA |
| CN111304372A (en) * | 2020-04-29 | 2020-06-19 | 圣湘生物科技股份有限公司 | Composition for detecting 2019 novel coronavirus mutation, application and kit |
| CN111334583A (en) * | 2020-01-19 | 2020-06-26 | 中国检验检疫科学研究院 | Method for identifying aedes species based on PCR-MS mass spectrometry scanning |
| CN111394438A (en) * | 2020-01-19 | 2020-07-10 | 中国医学科学院病原生物学研究所 | Neisseria gonorrhoeae drug-resistant site multiple detection method |
| CN111445955A (en) * | 2020-04-10 | 2020-07-24 | 广州微远基因科技有限公司 | Novel coronavirus variation analysis method and application |
| CN111455062A (en) * | 2020-04-01 | 2020-07-28 | 中国人民解放军总医院 | Kit and platform for detecting susceptibility genes of novel coronavirus |
| CN111518960A (en) * | 2020-06-16 | 2020-08-11 | 河北省儿童医院 | Multi-RT-qPCR kit for coronavirus typing detection, primer probe composition and using method thereof |
-
2020
- 2020-08-27 CN CN202010874232.5A patent/CN112029906B/en active Active
Patent Citations (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105238853A (en) * | 2015-08-20 | 2016-01-13 | 复旦大学附属华山医院 | Method for simultaneously measuring gene polymorphism of tacrolimus action targets of person |
| CN111334583A (en) * | 2020-01-19 | 2020-06-26 | 中国检验检疫科学研究院 | Method for identifying aedes species based on PCR-MS mass spectrometry scanning |
| CN111394438A (en) * | 2020-01-19 | 2020-07-10 | 中国医学科学院病原生物学研究所 | Neisseria gonorrhoeae drug-resistant site multiple detection method |
| CN111139317A (en) * | 2020-03-13 | 2020-05-12 | 欧陆分析技术服务(苏州)有限公司 | Multiplex fluorescent quantitative PCR detection kit and detection method for SARS-COV-2 virus |
| CN111235316A (en) * | 2020-03-23 | 2020-06-05 | 中国检验检疫科学研究院 | Primer probe for identifying novel coronavirus and application of primer probe in triple fluorescence RPA |
| CN111455062A (en) * | 2020-04-01 | 2020-07-28 | 中国人民解放军总医院 | Kit and platform for detecting susceptibility genes of novel coronavirus |
| CN111445955A (en) * | 2020-04-10 | 2020-07-24 | 广州微远基因科技有限公司 | Novel coronavirus variation analysis method and application |
| CN111304372A (en) * | 2020-04-29 | 2020-06-19 | 圣湘生物科技股份有限公司 | Composition for detecting 2019 novel coronavirus mutation, application and kit |
| CN111518960A (en) * | 2020-06-16 | 2020-08-11 | 河北省儿童医院 | Multi-RT-qPCR kit for coronavirus typing detection, primer probe composition and using method thereof |
Non-Patent Citations (2)
| Title |
|---|
| Detection of SARS-CoV-2 in nasal swabs using MALDI-MS;Fabiane M Nachtigall et,al.;《Nat Biotechnol.》;20200730;第38卷(第10期);第1168–1173页 * |
| Genome-wide analysis of Indian SARS-CoV-2 genomes for the identification of genetic mutation and SNP;Indrajit Saha et,al.;《Infection, Genetics and Evolution》;20200711;第85卷;104457 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN112029906A (en) | 2020-12-04 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN112029906B (en) | Two-dimensional code detection method for distinguishing SARS-CoV and SARS-CoV2 virus based on SNP | |
| CN113215312A (en) | Coronavirus SARS-CoV-2 digital PCR multiple detection kit and its application | |
| US20120183952A1 (en) | Compositions for use in identification of caliciviruses | |
| CN115323075A (en) | RT-RAA primer probe set and kit for detecting infectious bronchitis viruses and genotyping and application of RT-RAA primer probe set and kit | |
| CN111235251A (en) | Kit for detecting gene of nitrendipine individualized medication guidance of antihypertensive drug | |
| CN111235252A (en) | Method for distinguishing individual medication of nitrendipine by mass spectrometry through detecting product | |
| CN111187824A (en) | Nicholol medication guide gene detection kit for antihypertensive drug | |
| CN111961763A (en) | Novel gene chip for detecting coronavirus | |
| CN111235255A (en) | Method for distinguishing individual medication of nitrendipine by using primer composition through mass spectrometry | |
| CN111235254A (en) | Primer composition for distinguishing nitrendipine individualized medication type | |
| CN111187815A (en) | Method for distinguishing individual administration of losartan by detecting product and performing mass spectrometry | |
| CN111235253A (en) | Detection product for distinguishing nitrendipine individualized medication type | |
| CN111206087A (en) | Method for distinguishing trandolapril personalized medicine by using primer composition to perform mass spectrometry | |
| CN111206083A (en) | Detection kit for medication guidance gene of telmisartan medicament for reducing hypertension | |
| CN111206086A (en) | Method for distinguishing trandolapril personalized medicine by mass spectrometry through product detection | |
| CN111206085A (en) | Trandolapril medication guide gene detection kit for antihypertensive drug | |
| CN111206089A (en) | Primer composition for distinguishing trandolapril individual medicine type | |
| CN111206088A (en) | Detection product for distinguishing trandolapril individual medication type | |
| CN111100914A (en) | Method for carrying out perindopril personalized medication guidance by detecting product | |
| CN111172266A (en) | Kit for detecting drug-using guide gene of antihypertensive drug verapamil | |
| CN114540497B (en) | Marker for bladder cancer screening, probe composition and application thereof | |
| CN113913551B (en) | Sequencing primer, detection method and kit for dengue virus typing | |
| CN114231635B (en) | Marker and probe composition for lung cancer screening and application thereof | |
| CN114959120A (en) | Primer probe set and kit for detecting chicken infectious anemia virus by RAA fluorescence method and application of primer probe set and kit | |
| CN114807432A (en) | Kit and method for rapidly detecting novel coronavirus and Delta mutant strain thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |