CN113604609A - Primer combination for detecting SARS-CoV-2 and D614G mutant strain and application thereof - Google Patents
Primer combination for detecting SARS-CoV-2 and D614G mutant strain and application thereof Download PDFInfo
- Publication number
- CN113604609A CN113604609A CN202110900752.3A CN202110900752A CN113604609A CN 113604609 A CN113604609 A CN 113604609A CN 202110900752 A CN202110900752 A CN 202110900752A CN 113604609 A CN113604609 A CN 113604609A
- Authority
- CN
- China
- Prior art keywords
- cov
- sars
- detecting
- assisting
- preparing
- 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.)
- Granted
Links
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
- C12Q1/686—Polymerase chain reaction [PCR]
-
- 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)
- Chemical Kinetics & Catalysis (AREA)
- Biotechnology (AREA)
- Microbiology (AREA)
- Molecular Biology (AREA)
- Analytical Chemistry (AREA)
- Physics & Mathematics (AREA)
- Biophysics (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Engineering & Computer Science (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Genetics & Genomics (AREA)
- Virology (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention discloses a primer combination for detecting SARS-CoV-2 and D614G mutant strain and application thereof. The primer combination for detecting SARS-CoV-2 and its D614G mutant strain consists of 15 single-stranded DNAs shown in sequence 1-15 in the sequence table. The primer combination of the invention has strong specificity and high sensitivity when being used for detecting the novel coronavirus, the sample detection limit is 1-10 copy number/reaction, and the primer combination does not have cross reaction with other 6 kinds of coronavirus infecting human, thereby obviously reducing false negative results. The primer combination can be used for large-scale rapid in-vitro qualitative detection of novel coronavirus-infected suspected pneumonia cases, suspected aggregated venereal disease cases, and novel coronavirus genes in nasopharyngeal swabs, sputum, alveolar lavage fluid and other samples of patients needing novel coronavirus infection diagnosis or differential diagnosis, and has good application prospect.
Description
Technical Field
The invention relates to a primer combination for detecting SARS-CoV-2 and D614G mutant strain and application thereof, belonging to the field of biomedicine.
Background
Coronavirus (HCoV) causes respiratory and gastrointestinal infections mainly, and is genetically classified into four genera: alpha coronavirus, beta coronavirus, gamma coronavirus, and delta coronavirus. Six human-infecting coronaviruses have been identified to date including HCoV-NL63 and HCoV-229E of the genus alphacoronavirus and HCoV-OC43, HCoV-HKU1 of the genus betacoronavirus, Severe acute respiratory syndrome coronavirus (SARS-CoV) and middle east respiratory syndrome coronavirus (MERS-CoV). On 11.2.2020, the World Health Organization (WHO) named the 2019 novel coronavirus as COVID-19, and the International Committee for viral Classification (ICTV) named Severe acute respiratory syndrome coronavirus 2 (SARS-Cov-2). SARS-CoV-2 is a human-historically identified seventh human-infectable HCoV, whose whole genome has 76% sequence homology with SARS-CoV, compared to the other 5 coronavirus sequences.
The self-new coronavirus SARS-CoV-2 is spread in large area and has gradual variety, high infectivity and serious harm. The D614G mutation refers to the mutation of amino acid position D (aspartic acid) of 614 on the S protein of SARS-CoV-2 to G (glycine). The strain carrying the D614G mutation accounted for less than 10% of the global neo-coronavirus sequencing sequences at an early stage. After 4 months of spread transmission, the gene becomes the main genotype of virus transmission.
When the epidemic situation outbreak occurs in a large area, the method can quickly and accurately detect and diagnose infected persons or carriers, has vital function and significance for epidemic situation prevention and control, and can further classify SARS-CoV-2 infected by confirmed cases, thereby being more beneficial to analyzing and comparing pathological features and tracing infection sources. Nucleic acid detection is a gold standard for determination of SARS-CoV-2. The most widely used detection method at present is the real-time fluorescence RT-PCR method. The existing PCR detection kit for detecting SARS-CoV-2 has a large amount of false negative and a large amount of positive in the practical application process, and the sample processing capability per day is limited, thus being not beneficial to clinical rapid and accurate large-scale case screening; the requirement on the sample is high, and the nucleic acid detection is negative when the three times of nucleic acid detection are carried out according to the standard requirement of clinical discharge; similar to other coronavirus sequences infecting human, the selection of target genes and specific sequences does not refer to other coronavirus sequences for specific selection, and the sensitivity and accuracy of primer probe detection are required to be improved; only confirmed cases can be diagnosed, and further typing of SARS-CoV-2 infected by the confirmed cases cannot be performed.
Therefore, a low-cost detection method with high specificity, high sensitivity, high throughput and relatively simple operation is urgently needed to detect, identify and type the novel coronavirus SARS-CoV-2.
Disclosure of Invention
The invention aims to solve the technical problem of how to detect novel coronavirus SARS-CoV-2 and further how to detect a D614G mutant strain, wherein the D614G mutant strain is SARS-CoV-2 in which a codon encoding the 614 th aspartic acid in an S gene is mutated from GAT to a codon GGT of glycine.
In order to solve the technical problems, the invention firstly provides a primer combination (marked as a primer combination 1), wherein the primer combination 1 consists of a primer pair respectively named as ORF1b-P, N1-P, N2-P, S-P and S (D614G) -P and an MPE primer respectively named as ORF1b-T, N1-T, N2-T, S-T and S (D614G) -T;
the ORF1b-P consists of single-stranded DNA named ORF1b-F and ORF1b-R, respectively, and the sequences of the ORF1b-F and the ORF1b-R are sequence 1 and sequence 2, respectively;
the N1-P is composed of single-stranded DNA with the names of N1-F and N1-R, and the sequences of the N1-F and the N1-R are sequences 4 and 5 respectively;
the N2-P is composed of single-stranded DNA with the names of N2-F and N2-R, and the sequences of the N2-F and the N2-R are sequences 7 and 8 respectively;
the S-P consists of single-stranded DNA with the names of S-F and S-R respectively, and the sequences of the S-F and the S-R are respectively sequences 10 and 11;
the S (D614G) -P consists of single-stranded DNA named S (D614G) -F and S (D614G) -R, respectively, the sequences of the S (D614G) -F and the S (D614G) -R are sequences 13 and 14, respectively;
the ORF1b-T is a single-stranded DNA shown in a sequence 3 in a sequence table;
the N1-T is a single-stranded DNA shown in a sequence 6 in a sequence table;
the N2-T is a single-stranded DNA shown in a sequence 9 in a sequence table;
the S-T is single-stranded DNA shown as a sequence 12 in a sequence table;
and the S (D614G) -T is single-stranded DNA shown as a sequence 15 in the sequence table.
In the primer set 1, the number of moles of the ORF1b-F, the ORF1b-R, the N1-F, the N1-R, the N2-F, the N2-R, the S-F, the S-R, the S (D614G) -F and the S (D614G) -R may be equal to one another.
In the primer combination 1, the molar ratio of ORF1b-T, N1-T, N2-T, S-T and S (D614G) -T can be 13.07: 8.72: 11.09: 9.99: 11.99.
the primer combination 1 can have any one of the following purposes:
x1, detecting or assisting in detecting SARS-CoV-2;
x2, preparing or auxiliary detecting SARS-CoV-2 product;
x3, detecting or assisting to detect whether the sample to be detected contains SARS-CoV-2;
x4, preparing and detecting or detecting in an auxiliary way whether the sample to be detected contains SARS-CoV-2 products;
x5, detecting or assisting in detecting the D614G mutant strain of SARS-CoV-2;
x6, preparing D614G mutant strain product for detecting or assisting in detecting SARS-CoV-2;
x7, detecting or detecting in an auxiliary way whether the sample to be detected contains the D614G mutant strain of SARS-CoV-2;
x8, preparing a D614G mutant strain product for detecting or assisting in detecting whether the sample to be detected contains SARS-CoV-2;
x9, detecting or assisting to detect whether SARS-CoV-2 generates D614G mutation;
x10, preparing a product for detecting or assisting in detecting whether SARS-CoV-2 generates D614G mutation;
x11, diagnosis or auxiliary diagnosis of SARS-CoV-2 caused disease;
x12, preparing a product for diagnosing or assisting in diagnosing diseases caused by SARS-CoV-2;
x13, diagnosis or auxiliary diagnosis of diseases caused by D614G mutant strain of SARS-CoV-2;
x14, preparing a product for diagnosing or assisting in diagnosing diseases caused by the D614G mutant strain of SARS-CoV-2;
x15, screening or auxiliary screening of SARS-CoV-2 infected patients;
x16, preparing products for screening or auxiliary screening SARS-CoV-2 infection patients;
x17, screening or auxiliary screening for SARS-CoV-2D 614G mutant infected patients;
x18, preparing D614G mutant infected patient products for screening or auxiliary screening SARS-CoV-2.
The invention also provides another primer combination (marked as a primer combination 2), wherein the primer combination 2 consists of a primer pair named as S (D614G) -P and an MPE primer named as S (D614G) -T;
the S (D614G) -P consists of single-stranded DNA named S (D614G) -F and S (D614G) -R, respectively, the sequences of the S (D614G) -F and the S (D614G) -R are sequences 13 and 14, respectively;
and the S (D614G) -T is single-stranded DNA shown as a sequence 15 in the sequence table.
In the primer set 2, the number of moles of S (D614G) -F and S (D614G) -R may be equal to each other.
The primer combination 2 has any one of the following purposes:
y1, D614G mutant for detecting or assisting in detecting SARS-CoV-2;
y2, preparing D614G mutant strain product for detecting or assisting in detecting SARS-CoV-2;
y3, detecting or detecting the D614G mutant strain of SARS-CoV-2 in the sample;
y4, preparing a D614G mutant strain product for detecting or assisting in detecting whether the sample to be detected contains SARS-CoV-2;
y5, detecting or assisting to detect whether SARS-CoV-2 generates D614G mutation;
y6, preparing a product for detecting or assisting in detecting whether the SARS-CoV-2 generates D614G mutation;
y7, diagnosis or auxiliary diagnosis of diseases caused by D614G mutant strain of SARS-CoV-2;
y8, preparing a product for diagnosing or assisting in diagnosing diseases caused by the D614G mutant strain of SARS-CoV-2;
y9, screening or auxiliary screening for SARS-CoV-2D 614G mutant infected patients;
y10, preparing D614G mutant infected patient product for screening or auxiliary screening SARS-CoV-2.
The invention also provides a kit, which contains the primer combination 1 or the primer combination 2.
The kit may also contain other reagents required for performing PCR amplification and/or other reagents required for mass extension.
The other reagents required for PCR amplification and the other reagents required for mass extension can be both reagents in SNP typing kits (QT-SJ09-SNPs) (thawing Biotechnology (Qingdao) Ltd.), such as A1 component: PCR premix solution, SAP enzyme, SAP buffer solution, MPE enzyme, MPE buffer solution, E-ddNTPmix, matrix solution, resin, Oligoplate.
The kit may have any of the following uses:
x1, detecting or assisting in detecting SARS-CoV-2;
x2, preparing or auxiliary detecting SARS-CoV-2 product;
x3, detecting or assisting to detect whether the sample to be detected contains SARS-CoV-2;
x4, preparing and detecting or detecting in an auxiliary way whether the sample to be detected contains SARS-CoV-2 products;
x5, detecting or assisting in detecting the D614G mutant strain of SARS-CoV-2;
x6, preparing D614G mutant strain product for detecting or assisting in detecting SARS-CoV-2;
x7, detecting or detecting in an auxiliary way whether the sample to be detected contains the D614G mutant strain of SARS-CoV-2;
x8, preparing a D614G mutant strain product for detecting or assisting in detecting whether the sample to be detected contains SARS-CoV-2;
x9, detecting or assisting to detect whether SARS-CoV-2 generates D614G mutation;
x10, preparing a product for detecting or assisting in detecting whether SARS-CoV-2 generates D614G mutation;
x11, diagnosis or auxiliary diagnosis of SARS-CoV-2 caused disease;
x12, preparing a product for diagnosing or assisting in diagnosing diseases caused by SARS-CoV-2;
x13, diagnosis or auxiliary diagnosis of diseases caused by D614G mutant strain of SARS-CoV-2;
x14, preparing a product for diagnosing or assisting in diagnosing diseases caused by the D614G mutant strain of SARS-CoV-2;
x15, screening or auxiliary screening of SARS-CoV-2 infected patients;
x16, preparing products for screening or auxiliary screening SARS-CoV-2 infection patients;
x17, screening or auxiliary screening for SARS-CoV-2D 614G mutant infected patients;
x18, preparing D614G mutant infected patient products for screening or auxiliary screening SARS-CoV-2.
The following applications of the primer combination 1 or the primer combination 2 also belong to the protection scope of the invention:
x1, detecting or assisting in detecting SARS-CoV-2;
x2, preparing or auxiliary detecting SARS-CoV-2 product;
x3, detecting or assisting to detect whether the sample to be detected contains SARS-CoV-2;
x4, preparing and detecting or detecting in an auxiliary way whether the sample to be detected contains SARS-CoV-2 products;
x5, detecting or assisting in detecting the D614G mutant strain of SARS-CoV-2;
x6, preparing D614G mutant strain product for detecting or assisting in detecting SARS-CoV-2;
x7, detecting or detecting in an auxiliary way whether the sample to be detected contains the D614G mutant strain of SARS-CoV-2;
x8, preparing a D614G mutant strain product for detecting or assisting in detecting whether the sample to be detected contains SARS-CoV-2;
x9, detecting or assisting to detect whether SARS-CoV-2 generates D614G mutation;
x10, preparing a product for detecting or assisting in detecting whether SARS-CoV-2 generates D614G mutation;
x11, diagnosis or auxiliary diagnosis of SARS-CoV-2 caused disease;
x12, preparing a product for diagnosing or assisting in diagnosing diseases caused by SARS-CoV-2;
x13, diagnosis or auxiliary diagnosis of diseases caused by D614G mutant strain of SARS-CoV-2;
x14, preparing a product for diagnosing or assisting in diagnosing diseases caused by the D614G mutant strain of SARS-CoV-2;
x15, screening or auxiliary screening of SARS-CoV-2 infected patients;
x16, preparing products for screening or auxiliary screening SARS-CoV-2 infection patients;
x17, screening or auxiliary screening for SARS-CoV-2D 614G mutant infected patients;
x18, preparing D614G mutant infected patient products for screening or auxiliary screening SARS-CoV-2.
Any one of the following applications of the kit also belongs to the protection scope of the invention:
x1, detecting or assisting in detecting SARS-CoV-2;
x2, preparing or auxiliary detecting SARS-CoV-2 product;
x3, detecting or assisting to detect whether the sample to be detected contains SARS-CoV-2;
x4, preparing and detecting or detecting in an auxiliary way whether the sample to be detected contains SARS-CoV-2 products;
x5, detecting or assisting in detecting the D614G mutant strain of SARS-CoV-2;
x6, preparing D614G mutant strain product for detecting or assisting in detecting SARS-CoV-2;
x7, detecting or detecting in an auxiliary way whether the sample to be detected contains the D614G mutant strain of SARS-CoV-2;
x8, preparing a D614G mutant strain product for detecting or assisting in detecting whether the sample to be detected contains SARS-CoV-2;
x9, detecting or assisting to detect whether SARS-CoV-2 generates D614G mutation;
x10, preparing a product for detecting or assisting in detecting whether SARS-CoV-2 generates D614G mutation;
x11, diagnosis or auxiliary diagnosis of SARS-CoV-2 caused disease;
x12, preparing a product for diagnosing or assisting in diagnosing diseases caused by SARS-CoV-2;
x13, diagnosis or auxiliary diagnosis of diseases caused by D614G mutant strain of SARS-CoV-2;
x14, preparing a product for diagnosing or assisting in diagnosing diseases caused by the D614G mutant strain of SARS-CoV-2;
x15, screening or auxiliary screening of SARS-CoV-2 infected patients;
x16, preparing products for screening or auxiliary screening SARS-CoV-2 infection patients;
x17, screening or auxiliary screening for SARS-CoV-2D 614G mutant infected patients;
x18, preparing D614G mutant infected patient products for screening or auxiliary screening SARS-CoV-2.
The invention combines the multiplex PCR and MALDI-TOFMS method to obtain a group of primer combination which can detect SARS-CoV-2 and D614G mutant strain, the specificity of the primer combination for detecting novel coronavirus is strong, the sensitivity is high, the sample detection limit is 1-10 copy number/reaction, no cross reaction occurs with other 6 kinds of coronavirus infecting human, and the false negative result can be obviously reduced. The invention has less requirements on samples and low detection cost, can simultaneously detect hundreds of samples and realizes high-flux detection in a short time. Besides, it can further classify SARS-CoV-2 infected by confirmed case, and is helpful for analyzing and comparing pathological characteristics and tracing infection source. The primer combination can be used for large-scale rapid in-vitro qualitative detection of novel coronavirus-infected suspected pneumonia cases, suspected aggregated venereal disease cases, and novel coronavirus genes in nasopharyngeal swabs, sputum, alveolar lavage fluid and other samples of patients needing novel coronavirus infection diagnosis or differential diagnosis, and has good application prospect.
Detailed Description
The present invention is described in further detail below with reference to specific embodiments, which are given for the purpose of illustration only and are not intended to limit the scope of the invention. The examples provided below serve as a guide for further modifications by a person skilled in the art and do not constitute a limitation of the invention in any way.
The experimental procedures in the following examples, unless otherwise indicated, are conventional and are carried out according to the techniques or conditions described in the literature in the field or according to the instructions of the products. Materials, reagents, instruments and the like used in the following examples are commercially available unless otherwise specified. The quantitative tests in the following examples, all set up three replicates and the results averaged. In the following examples, unless otherwise specified, the 1 st position of each nucleotide sequence in the sequence listing is the 5 'terminal nucleotide of the corresponding DNA, and the last position is the 3' terminal nucleotide of the corresponding DNA.
SNP typing kit (QT-SJ09-SNPs) from GloZhi Biotechnology (Qingdao) Ltd, which contains the following reagents:
example 1 preparation of primer combination for detection of novel coronavirus
1. Preparation of primer combinations
This example prepared a primer set for detecting a novel coronavirus consisting of a primer pair named ORF1b-P, N1-P, N2-P, S-P and S (D614G) -P, respectively, and MPE primers named ORF1b-T, N1-T, N2-T, S-T and S (D614G) -T, respectively. ORF1b-P consists of single-stranded DNA named ORF1b-F and ORF1b-R, respectively, and the sequences are sequences 1 and 2, respectively; N1-P is composed of single-stranded DNA with the names of N1-F and N1-R, and the sequences are respectively sequence 4 and 5; N2-P is composed of single-stranded DNA with the names of N2-F and N2-R, and the sequences are respectively sequence 7 and 8; S-P is composed of single-stranded DNA with the names of S-F and S-R respectively, and the sequences of the single-stranded DNA are respectively 10 and 11; s (D614G) -P is composed of single-stranded DNA named S (D614G) -F and S (D614G) -R, respectively, and the sequences are sequences 13 and 14, respectively; the sequences of ORF1b-T, N1-T, N2-T, S-T and S (D614G) -T are sequences 3, 6, 9, 12, 15, respectively. The sequence information of each primer is shown in Table 1.
In the primer combination, the mole numbers of ORF1b-F, ORF1b-R, N1-F, N1-R, N2-F, N2-R, S-F, S-R, S (D614G) -F, S (D614G) -R are equal;
the molar ratio of ORF1b-T, N1-T, N2-T, S-T and S (D614G) -T is 13.07: 8.72: 11.09: 9.99: 11.99.
TABLE 1 primer combination information
In Table 1, S (D614G) is a codon "GGT" in which the codon encoding aspartic acid at position 614 in the S gene is mutated from "GAT" to glycine, and SARS-CoV-2 containing the mutation is a D614G mutant.
The theoretical values of MPE primer molecular weight and MPE product molecular weight are shown in Table 2, and there is a deviation of about + -2 Da in mass in the actual detection process.
TABLE 2 theoretical values of MPE primer molecular weight and MPE product molecular weight
2. Preparation of recombinant plasmid
Recombinant plasmids of DNA fragments of HCoV-229E, HCoV-OC43, HCoV-NL63, HCoV-HKU1, SARS-CoV, MERS-CoV, SARS-CoV-2 were constructed, wherein HCoV-HKU1 synthesized two plasmids, pUC57-HCoV-HKU1-1 and pUC57-HCoV-HKU 1-2, respectively, according to the selection of mutation frequency because of the relatively large number of mutation sites of the conserved sequences within the species.
The recombinant plasmids pUC57-SARS-CoV-2 and pUC57-SARS-CoV-2 containing the DNA fragment of SARS-CoV-2 were constructed by replacing the DNA fragment between the SacI and SalI recognition sequences of the plasmid pUC57 with the DNA fragment shown in sequence 16 of the sequence Listing.
A recombinant plasmid pUC57-HCoV-229E containing the DNA fragment of HCoV-229E, and pUC57-HCoV-229E were constructed by replacing the DNA fragment between the BamHI and SalI recognition sequences of the pUC57 plasmid with the DNA fragment shown in sequence 17 of the sequence Listing.
Construction of recombinant plasmid pUC57-HCoV-OC43 and pUC57-HCoV-OC43 each containing the DNA fragment of HCoV-OC43 was a recombinant plasmid obtained by replacing the DNA fragment between the XbaI and SalI recognition sequences of pUC57 plasmid with the DNA fragment shown in sequence 18 of the sequence Listing.
Construction of recombinant plasmids pUC57-HCoV-NL63 and pUC57-HCoV-NL63 each containing a DNA fragment of HCoV-NL63 were recombinant plasmids obtained by replacing a DNA fragment between BamHI and SalI recognition sequences of a pUC57 plasmid with a DNA fragment shown by sequence 19 in the sequence Listing.
Construction of pUC57-HCoV-HKU1-1 and pUC57-HCoV-HKU1-1 are recombinant plasmids obtained by replacing the DNA fragment between the XbaI and SalI recognition sequences of the pUC57 plasmid with the DNA fragment shown as sequence 20 in the sequence Listing.
Construction of pUC57-HCoV-HKU 1-2 and pUC57-HCoV-HKU 1-2 is a recombinant plasmid obtained by replacing the DNA fragment between the BamHI and SalI recognition sequences of the pUC57 plasmid with the DNA fragment shown in sequence 21 of the sequence Listing.
A recombinant plasmid pUC57-SARS-CoV containing the DNA fragment of SARS-CoV was constructed, and pUC57-SARS-CoV was a recombinant plasmid obtained by replacing the DNA fragment between the BamHI and SalI recognition sequences of the plasmid pUC57 with the DNA fragment shown in sequence 22 of the sequence Listing.
Constructing a recombinant plasmid containing a DNA fragment of MERS-CoV, wherein pUC57-MERS-CoV is obtained by replacing the DNA fragment between BamHI and SalI recognition sequences of the pUC57 plasmid with the DNA fragment shown in the sequence 23 in the sequence table.
Example 2 method for detecting novel coronavirus Using the primer combination of example 1
The amplification efficiency of the primer combination of example 1 was measured using an SNP typing kit (QT-SJ09-SNPs) by the following steps:
1. preparation of primer mixture
Preparing a primer mixed solution: primer pairs ORF1b-P, N1-P, N2-P, S-P and S (D614G) -P of example 1 were dissolved in RNase-free deionized water to obtain a primer mixture, in which the concentrations of ORF1b-F, ORF1b-R, N1-F, N1-R, N2-F, N2-R, S-F, S-R, S (D614G) -F and S (D614G) -R were 0.5. mu.M.
2. Multiplex PCR amplification
Multiplex PCR amplification was performed using the primer mixture of step 1, and the DNA template was pUC57-SARS-CoV-2 of example 1, as follows:
| components | Volume/. mu.l |
| PCR premix | 2 |
| Deionized water | 1 |
| Primer mixture | 1 |
| DNA template (concentration 10)2copies/μL) | 1 |
| Total of | 5 |
RNase-free ddH2O was used as a negative control in place of the DNA template. Two replicates were set for each system.
Performing multiplex PCR amplification on the obtained system under the following conditions to obtain a multiplex PCR product: 15min at 95 ℃; 15s at 95 ℃, 30s at 59 ℃, 30s at 72 ℃ and 30 cycles; 10min at 60 ℃; keeping at 4 ℃.
3. SAP digestion
The reaction system adds the following components to the multiplex PCR product of step 2:
| components | Volume/. mu.l |
| Deionized water | 1.53 |
| SAP buffer | 0.17 |
| SAP enzymes | 0.3 |
| Total of | 2 |
Reacting the obtained reaction system under the following conditions to obtain SAP digestion products: 40min at 37 ℃; 5min at 85 ℃; keeping at 4 ℃.
4. Mass extension (MPE)
The reaction system added the following components to the SAP digest product of step 3:
| components | Volume/. mu.l |
| E-ddNTPmix | 1 |
| MEP buffer | 1.4 |
| MEP enzymes | 0.6 |
| MPE primers | 1 |
| Total of | 4 |
The MPE primers are ORF1b-T, N1-T, N2-T, S-T and S (D614G) -T, and the concentrations of ORF1b-T, N1-T, N2-T, S-T and S (D614G) -T in the system are respectively 13.07 mu M, 8.72 mu M, 11.09 mu M, 9.99 mu M and 11.99 mu M. The obtained reaction system is reacted under the following conditions to obtain a mass extension product:
5. resin desalination
[ step 4 ] after completion of the reaction, 14. mu.l of RNase-free ddH2O was added to each reaction well.
And lightly turning the eight rows of tubes filled with the resin to buckle the eight rows of tubes on the sample plate, so as to ensure that the resin holes are aligned with each hole of the sample. The resin tube was then tapped to drop the resin into the wells of the sample plate.
And thirdly, using a palm centrifuge for instantaneous centrifugation to avoid the resin from being attached to the pipe wall.
And fourthly, placing the sample plate with the resin in an overturning blending instrument, and blending for 30min at 20 rpm.
Fifthly, after the mixing is finished, centrifuging the sample at 2000rpm for 1min, and collecting the supernatant to be tested.
6. Nucleic acid mass spectrometry to be detected
Mixing the matrix liquid fully and carrying out ultrasonic treatment for 2 min. And (3) putting 1 mu l of matrix at the center of the target spot, standing at room temperature for 4-5 min, and naturally drying.
② sucking 0.5 mul of the supernatant obtained in the step 5 on the substrate, drying and crystallizing to be tested.
And thirdly, performing on-machine detection by adopting a QuandTOF I mass spectrometer. And 3 target plates are arranged on each system for on-machine detection.
And (3) related setting and parameters during computer-on:
1) instrument parameter setting
Collecting machine types: QuandTOF I
An acquisition mode: linear positive ion mode
Accelerate Voltage:20kV
Mass Range:3000-11000Da
Laser Frequency:3000Hz
Shots/Spectrum:800
Laser energy 30uJ
2) Instrumental molecular weight calibration
The instrument was calibrated using calibrators (4k-10kDa, Tokyo Biotechnology (Qingdao) Ltd.) of molecular weights 4550.0Da, 5478.6Da, 6332.2Da, 7717.0Da, 9507.2Da, respectively.
Each primer can be specifically amplified, and the results are shown in Table 3.
TABLE 3 detection results of 5 sites in pUC57-SARS-CoV-2
Specifically, the method for determining whether the sample to be tested contains SARS-CoV-2 according to the detection result of the primer combination in example 1 is as follows:
if the MPE product signal peak signal-to-noise ratio of at least one of the N1, N2, ORF1b and S genes is greater than 4, the sample to be detected contains SARS-CoV-2 and is not a D614G mutant strain; if the MPE product signal peak signal-to-noise ratios of N1, N2, ORF1b and S gene are all less than or equal to 4, the sample to be detected does not contain SARS-CoV-2.
Furthermore, when the sample to be tested contains SARS-CoV-2, if the MPE product of S (D614G) is amplified to G and the signal peak signal-to-noise ratio is greater than 4, the SARS-CoV-2 in the sample to be tested is D614G mutant strain.
Specificity of primer combinations of example 3 and example 1
pUC57-SARS-CoV-2, pUC57-HCoV-229E, pUC57-HCoV-OC43, pUC57-HCoV-NL63, pUC57-HCoV-HKU1-1, pUC57-HCoV-HKU 1-2, pUC57-SARS-CoV and pUC57-MERS-CoV of example 1 were subjected to RNase-free ddH reaction, respectively2Dissolving the plasmid to obtain the plasmid with the concentration of 105The eight plasmid solutions, pUC57-SARS-CoV-2 solution, pUC57-HCoV-229E solution, pUC57-HCoV-OC43 solution, pUC57-HCoV-NL63 solution, pUC57-HCoV-HKU1-1 solution, pUC57-HCoV-HKU 1-2 solution, pUC57-SARS-CoV solution and pUC57-MERS-CoV solution, were copied/. mu.l.
The specificity of the primer combination of example 1 was examined by replacing the DNA templates with the above eight plasmid solutions by the methods of 2 to 6 in example 2, respectively, and the other steps were not changed.
As shown in Table 4, the primer combination of example 1 showed excellent specificity, no plasmids containing other coronavirus fragments were amplified, 5 target sites of SARS-CoV-2 were specifically amplified and detected, and SARS-CoV-2 was detected without interference from other coronaviruses.
TABLE 4 specific detection of SARS-CoV-2 primer combination
Sensitivity of primer combinations of examples 4 and 1
pUC57-SARS-CoV-2 of example 1 was dissolved with RNase-free ddH2O and diluted with RNase-free ddH2O to give concentrations of 100 copies/. mu.l and 10 copies/. mu.l, respectively1Copy/. mu.l, 102Copy/. mu.l, 103Copy/. mu.l, 104Copy/. mu.l, 105Copies/. mu.l plasmid dilution.
The sensitivity of the primer combination of example 1 was measured by replacing the DNA template with each of the above-mentioned plasmid dilutions by the methods of examples 2 to 6, setting the number of cycles of multiplex PCR amplification to 30, 35, 40, and 45 cycles, and keeping the other steps unchanged. One plasmid dilution per system, 2 replicate wells per plasmid dilution per multiplex PCR cycle.
The results of the assay showed that the number of cycles had no effect on sensitivity. The results for 45 cycles are shown in table 5 with a minimum detection limit of 10 copies/reaction.
When the plasmid concentration of the added plasmid diluent is 10 copies/mu l, 5 target targets can be detected completely.
TABLE 545 amplification of different concentrations of pUC57-SARS-CoV-2 at cycles
| Plasmid concentration (copies/. mu.L) -repeats | N1 | N2 | ORF1b | S | S(D614G) |
| 100Repetition of 1 | No amplification | No amplification | No amplification | No amplification | No amplification |
| 100Repetition 2 | No amplification | No amplification | No amplification | No amplification | No amplification |
| 101Repetition of 1 | G | G | A | G | A |
| 101Repetition 2 | G | G | A | G | A |
| 102Repetition of 1 | G | G | A | G | A |
| 102Repetition 2 | G | G | A | G | A |
| 103Repetition of 1 | G | G | A | G | A |
| 103Repetition 2 | G | G | A | G | A |
| 104Repetition of 1 | G | G | A | G | A |
| 104Repetition 2 | G | G | A | G | A |
| 105Repetition of 1 | G | G | A | G | A |
| 105Repetition 2 | G | G | A | G | A |
Example 5 and application of primer combination of example 1 to detection of actual sample
Pharyngeal swab clinical samples of 11 suspected SARS-CoV-2 infected cases were collected and numbered 1-11, respectively.
The samples were tested by whole genome sequencing as follows: 8 parts are negative (numbered 1-8) and 3 parts are positive (numbered 9-11). 2 of the 3 positives were further typed as D614G mutants (numbered 9-10).
The specific procedures for detecting clinical samples using the primer combinations of example 1 are as follows:
1. extracting total RNA of each throat swab clinical sample, and carrying out reverse transcription to obtain cDNA.
2. The DNA templates were replaced with cDNA from each of the above samples by the methods 2-6 of example 2, and 2 negative controls were set up for each sample in parallel (i.e., the DNA templates were replaced with deionized water).
The results are shown in tables 6 to 16:
as can be seen from Table 14, sample No. 9 was confirmed to be a positive sample by MPE occurring in 4 target genes of SARS-CoV-2, N1, N2, ORF1b and S, S (D614G) and having a signal-to-noise ratio > 4.
As is clear from Table 14, the result of MPE in the S gene (containing the D614G mutation site) in sample No. 9 was G, indicating that SARS-CoV-2 in this positive sample was D614G mutant.
As is clear from Table 15, MPE was found in the SARS-CoV-2 samples No. 10 in both of N1 and S, S (D614G)3 target genes, and these were confirmed to be positive samples.
As is clear from Table 15, the result of MPE in the S gene (containing the D614G mutation site) in sample No. 10 was G, indicating that SARS-CoV-2 in the positive sample was D614G mutant.
As is clear from Table 16, MPE was found in all of the N1, N2 and ORF1b 3 target genes of SARS-CoV-2 in sample No. 11, which confirmed that the sample was a positive sample.
As is clear from Table 16, the fact that MPE did not occur in the S gene (containing the D614G mutation site) in sample No. 11 indicates that SARS-CoV-2 in this positive sample is not the D614G mutant.
As is clear from tables 6 to 13, no MPE occurred in 5 target genes of SARS-CoV-2 in the clinical specimens No. 1 to No. 8, and all the specimens were negative.
This result is consistent with the results of gene sequencing.
Table 61 clinical sample test results
Table 72 clinical sample test results
Table 83 clinical sample test results
Table 94 clinical sample test results
Table 105 clinical sample test results
Table 116 clinical sample test results
Table 127 clinical sample test results
Table 138 clinical sample test results
Table 149 clinical sample test results
Test results of clinical specimens No. 1510 in Table
TABLE 1611 clinical specimen test results
The present invention has been described in detail above. It will be apparent to those skilled in the art that the invention can be practiced in a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the invention and without undue experimentation. While the invention has been described with reference to specific embodiments, it will be appreciated that the invention can be further modified. In general, this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains. The use of some of the essential features is possible within the scope of the claims attached below.
<110> military medical research institute of military science institute of people's liberation force of China
<120> primer combination for detecting SARS-CoV-2 and its D614G mutant strain and application thereof
<213> Artificial sequence (Artificial sequence)
<160> 23
<170> PatentIn version 3.5
<210> 1
<211> 21
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 1
tgctgtagat gctgctaaag c 21
<210> 2
<211> 20
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 2
gcctgaccag taccagtgtg 20
<210> 3
<211> 26
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 3
cccatcttaa cacaattagt gattgg 26
<210> 4
<211> 20
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 4
agaatggaga acgcagtggg 20
<210> 5
<211> 20
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 5
cggtgaacca agacgcagta 20
<210> 6
<211> 17
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 6
cgacgttgtt ttgatcg 17
<210> 7
<211> 20
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 7
caactccagg cagcagtagg 20
<210> 8
<211> 20
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 8
tgtcaagcag cagcaaagca 20
<210> 9
<211> 22
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 9
tctggctggc aatggcggtg at 22
<210> 10
<211> 21
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 10
acaggcacag gtgttcttac t 21
<210> 11
<211> 21
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 11
tggatcacgg acagcatcag t 21
<210> 12
<211> 20
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 12
ctcaatttgg cagagacatt 20
<210> 13
<211> 24
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 13
acttctaacc aggttgctgt tctt 24
<210> 14
<211> 24
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 14
cacgccaagt aggagtaagt tgat 24
<210> 15
<211> 24
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 15
ccagggactt ctgtgcagtt aaca 24
<210> 16
<211> 949
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 16
cttttgctgt agatgctgct aaagcttaca aagattatct agctagtggg ggacaaccaa 60
tcactaattg tgttaagatg ttgtgtacac acactggtac tggtcaggca ataacagtta 120
caccggaagc caatatggat caagaatcct ttggtggtgc atcgtgttgt ctgtactgcc 180
gttgccacat agatcatcca aatcctaaag gattttgtga cttaaaaggt aagtatgtac 240
aaatacctac aacttgtgct aatgaccctg tgggttttac acttaaaaac acagtctgta 300
ccgtctgcgg tatgtggaaa ggttatggct gtagttgtga tcaactccgc gaacccatgc 360
ttcagtcagc tgatgcacaa tcgtttttat aatggacccc aaaatcagcg aaatgcaccc 420
cgcattacgt ttggtggacc ctcagattca actggcagta accagaatgg agaacgcagt 480
ggggcgcgat caaaacaacg tcggccccaa ggtttaccca ataatactgc gtcttggttc 540
accgctctca gttcaagaaa ttcaactcca ggcagcagta ggggaacttc tcctgctaga 600
atggctggca atggcggtga tgctgctctt gctttgctgc tgcttgacag attgaaccaa 660
ctgattacaa acattggccg caaattgcac aatttgcccc cagcgcttca gcgttcttcg 720
gaatgtcgcg cattgggttt aacaggcaca ggtgttctta ctgagtctaa caaaaagttt 780
ctgcctttcc aacaatttgg cagagacatt gctgacacta ctgatgctgt ccgtgatcca 840
cagaccaaat acttctaacc aggttgctgt tctttatcag gatgttaact gcacagaagt 900
ccctgttgct attcatgcag atcaacttac tcctacttgg cgtgtttat 949
<210> 17
<211> 208
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 17
aggttacggt gttaggcgca agaattcaga accagagata ccacacttca atcaaaagct 60
cccaaatggt gttactgttg ttgaagaacc tgactcccgt gctccttccc ggttatggac 120
cacgagcagt ccatgtataa cttacttaaa ggctgtaatg ctgttgctaa gcatgatttc 180
tttacttggc atgagggcag aaccattt 208
<210> 18
<211> 188
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 18
cgctagcaac caggctgatg tcaatacccc ggctgacatt gtcgatcggg acccaagtag 60
cgatgaggct attccgacta ggtttccgcc tggctgcagc cccaggatgt ggtgttgcta 120
tagcagattc ttattattct tatatcatgc ctatgctgac catgtgtcat gcattggatt 180
gcgaattg 188
<210> 19
<211> 246
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 19
atcttgttgc tgctgttact ttggctttaa agaacttagg ttttgataac cagtcgaagt 60
cacctagttc ttctggtact tccactccta agaaacctaa taagcctctt tctcaaccca 120
gggctgataa gccttcgttt ttatagatgg tgttccactt gttacaactg ctggttatca 180
ttttaagcaa ttaggtttgg tttggaataa agatgttaac acacactcag ttaggttgac 240
aattac 246
<210> 20
<211> 181
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 20
atactcccgg tcatcatgct ggaagtagaa gctcctctgg aaatcgttca ggaatcctca 60
agaaaacttc ttgggttgac caatctgagc gaaacacacc gctatcgttt gtctcttaaa 120
gacttacttc tttatgcagc agatcctgct atgcatgttg catctgctag tgctctgctt 180
g 181
<210> 21
<211> 186
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 21
gtcttatact cccggtcatt atgctggaag tagaagctcc tctggaaatc gttcaggaat 60
cctcaagaaa acttcttggg ctgaccaatc tgagcgaaac acaccgttat cgtttgtctc 120
ttaaagattt acttctttat gcagcagatc ctgctatgca cgttgcatct gctagtgctc 180
tgcttg 186
<210> 22
<211> 280
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 22
gactgatgaa gctcagcctt tgccgcagag acaaaagaag cagcccactg tgactcttct 60
tcctgcggct gacatggatg atttctccca ctagccatcc ttactgcgct tcgattgtgt 120
gcgtactgct gcaatattgt taacgtgagt ttagtaaaac caacggttta cgtctactcg 180
cgtgttaacc tttttagaaa cgcccgtaat ggtgttttaa taacagaagg ttcagtcaaa 240
ggtctaacac cttcaaaggg accagcacaa gctagcgtca 280
<210> 23
<211> 437
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 23
gggtattggc ggagacagga cagaaaaatt aataccggga atggaattaa gcaactggct 60
cccaggtggt acttctacta cactggaact ggacccgaag cagcactccc attccgggct 120
ctataacact cttggtgtgt atggctttcc ttacggctac tagattatgt gtgcaatgta 180
tgacaggctt caataccctg ttagttcagc ccgcattata acttggagaa cgtgtacgcc 240
aagctatctt aaacactgtt aaattttgtg accacatggt caaggctggt ttagtcggtg 300
tgctcacact acgtgctgct cttcttgccg gttcatttga caaagtctat gatattggca 360
atcctaaagg aattcctatt gttgatgacc ctgtggttga ttggcattat tttgatgcac 420
agcccttgac caggaag 437
Claims (9)
1. A primer combination consisting of a primer pair named ORF1b-P, N1-P, N2-P, S-P and S (D614G) -P respectively and an MPE primer named ORF1b-T, N1-T, N2-T, S-T and S (D614G) -T respectively;
the ORF1b-P consists of single-stranded DNA named ORF1b-F and ORF1b-R, respectively, and the sequences of the ORF1b-F and the ORF1b-R are sequence 1 and sequence 2, respectively;
the N1-P is composed of single-stranded DNA with the names of N1-F and N1-R, and the sequences of the N1-F and the N1-R are sequences 4 and 5 respectively;
the N2-P is composed of single-stranded DNA with the names of N2-F and N2-R, and the sequences of the N2-F and the N2-R are sequences 7 and 8 respectively;
the S-P consists of single-stranded DNA with the names of S-F and S-R respectively, and the sequences of the S-F and the S-R are respectively sequences 10 and 11;
the S (D614G) -P consists of single-stranded DNA named S (D614G) -F and S (D614G) -R, respectively, the sequences of the S (D614G) -F and the S (D614G) -R are sequences 13 and 14, respectively;
the ORF1b-T is a single-stranded DNA shown in a sequence 3 in a sequence table;
the N1-T is a single-stranded DNA shown in a sequence 6 in a sequence table;
the N2-T is a single-stranded DNA shown in a sequence 9 in a sequence table;
the S-T is single-stranded DNA shown as a sequence 12 in a sequence table;
and the S (D614G) -T is single-stranded DNA shown as a sequence 15 in the sequence table.
2. The primer combination of claim 1, wherein: the primer combination has any one of the following purposes:
x1, detecting or assisting in detecting SARS-CoV-2;
x2, preparing or auxiliary detecting SARS-CoV-2 product;
x3, detecting or assisting to detect whether the sample to be detected contains SARS-CoV-2;
x4, preparing and detecting or detecting in an auxiliary way whether the sample to be detected contains SARS-CoV-2 products;
x5, detecting or assisting in detecting the D614G mutant strain of SARS-CoV-2;
x6, preparing D614G mutant strain product for detecting or assisting in detecting SARS-CoV-2;
x7, detecting or detecting in an auxiliary way whether the sample to be detected contains the D614G mutant strain of SARS-CoV-2;
x8, preparing a D614G mutant strain product for detecting or assisting in detecting whether the sample to be detected contains SARS-CoV-2;
x9, detecting or assisting to detect whether SARS-CoV-2 generates D614G mutation;
x10, preparing a product for detecting or assisting in detecting whether SARS-CoV-2 generates D614G mutation;
x11, diagnosis or auxiliary diagnosis of SARS-CoV-2 caused disease;
x12, preparing a product for diagnosing or assisting in diagnosing diseases caused by SARS-CoV-2;
x13, diagnosis or auxiliary diagnosis of diseases caused by D614G mutant strain of SARS-CoV-2;
x14, preparing a product for diagnosing or assisting in diagnosing diseases caused by the D614G mutant strain of SARS-CoV-2;
x15, screening or auxiliary screening of SARS-CoV-2 infected patients;
x16, preparing products for screening or auxiliary screening SARS-CoV-2 infection patients;
x17, screening or auxiliary screening for SARS-CoV-2D 614G mutant infected patients;
x18, preparing D614G mutant infected patient products for screening or auxiliary screening SARS-CoV-2.
3. A primer combination consisting of a primer pair named S (D614G) -P and an MPE primer named S (D614G) -T;
the S (D614G) -P consists of single-stranded DNA named S (D614G) -F and S (D614G) -R, respectively, the sequences of the S (D614G) -F and the S (D614G) -R are sequences 13 and 14, respectively;
and the S (D614G) -T is single-stranded DNA shown as a sequence 15 in the sequence table.
4. The primer combination of claim 3, wherein: the primer combination has any one of the following purposes:
y1, D614G mutant for detecting or assisting in detecting SARS-CoV-2;
y2, preparing D614G mutant strain product for detecting or assisting in detecting SARS-CoV-2;
y3, detecting or detecting the D614G mutant strain of SARS-CoV-2 in the sample;
y4, preparing a D614G mutant strain product for detecting or assisting in detecting whether the sample to be detected contains SARS-CoV-2;
y5, detecting or assisting to detect whether SARS-CoV-2 generates D614G mutation;
y6, preparing a product for detecting or assisting in detecting whether the SARS-CoV-2 generates D614G mutation;
y7, diagnosis or auxiliary diagnosis of diseases caused by D614G mutant strain of SARS-CoV-2;
y8, preparing a product for diagnosing or assisting in diagnosing diseases caused by the D614G mutant strain of SARS-CoV-2;
y9, screening or auxiliary screening for SARS-CoV-2D 614G mutant infected patients;
y10, preparing D614G mutant infected patient product for screening or auxiliary screening SARS-CoV-2.
5. A kit comprising the primer combination of claim 1 or 3.
6. The kit of claim 5, wherein: the kit may also contain other reagents required for performing PCR amplification and/or other reagents required for mass extension.
7. The kit according to claim 5 or 6, characterized in that: the kit has any one of the following uses:
x1, detecting or assisting in detecting SARS-CoV-2;
x2, preparing or auxiliary detecting SARS-CoV-2 product;
x3, detecting or assisting to detect whether the sample to be detected contains SARS-CoV-2;
x4, preparing and detecting or detecting in an auxiliary way whether the sample to be detected contains SARS-CoV-2 products;
x5, detecting or assisting in detecting the D614G mutant strain of SARS-CoV-2;
x6, preparing D614G mutant strain product for detecting or assisting in detecting SARS-CoV-2;
x7, detecting or detecting in an auxiliary way whether the sample to be detected contains the D614G mutant strain of SARS-CoV-2;
x8, preparing a D614G mutant strain product for detecting or assisting in detecting whether the sample to be detected contains SARS-CoV-2;
x9, detecting or assisting to detect whether SARS-CoV-2 generates D614G mutation;
x10, preparing a product for detecting or assisting in detecting whether SARS-CoV-2 generates D614G mutation;
x11, diagnosis or auxiliary diagnosis of SARS-CoV-2 caused disease;
x12, preparing a product for diagnosing or assisting in diagnosing diseases caused by SARS-CoV-2;
x13, diagnosis or auxiliary diagnosis of diseases caused by D614G mutant strain of SARS-CoV-2;
x14, preparing a product for diagnosing or assisting in diagnosing diseases caused by the D614G mutant strain of SARS-CoV-2;
x15, screening or auxiliary screening of SARS-CoV-2 infected patients;
x16, preparing products for screening or auxiliary screening SARS-CoV-2 infection patients;
x17, screening or auxiliary screening for SARS-CoV-2D 614G mutant infected patients;
x18, preparing D614G mutant infected patient products for screening or auxiliary screening SARS-CoV-2.
8. Use of any one of the primer combinations of claims 1-4 for:
x1, detecting or assisting in detecting SARS-CoV-2;
x2, preparing or auxiliary detecting SARS-CoV-2 product;
x3, detecting or assisting to detect whether the sample to be detected contains SARS-CoV-2;
x4, preparing and detecting or detecting in an auxiliary way whether the sample to be detected contains SARS-CoV-2 products;
x5, detecting or assisting in detecting the D614G mutant strain of SARS-CoV-2;
x6, preparing D614G mutant strain product for detecting or assisting in detecting SARS-CoV-2;
x7, detecting or detecting in an auxiliary way whether the sample to be detected contains the D614G mutant strain of SARS-CoV-2;
x8, preparing a D614G mutant strain product for detecting or assisting in detecting whether the sample to be detected contains SARS-CoV-2;
x9, detecting or assisting to detect whether SARS-CoV-2 generates D614G mutation;
x10, preparing a product for detecting or assisting in detecting whether SARS-CoV-2 generates D614G mutation;
x11, diagnosis or auxiliary diagnosis of SARS-CoV-2 caused disease;
x12, preparing a product for diagnosing or assisting in diagnosing diseases caused by SARS-CoV-2;
x13, diagnosis or auxiliary diagnosis of diseases caused by D614G mutant strain of SARS-CoV-2;
x14, preparing a product for diagnosing or assisting in diagnosing diseases caused by the D614G mutant strain of SARS-CoV-2;
x15, screening or auxiliary screening of SARS-CoV-2 infected patients;
x16, preparing products for screening or auxiliary screening SARS-CoV-2 infection patients;
x17, screening or auxiliary screening for SARS-CoV-2D 614G mutant infected patients;
x18, preparing D614G mutant infected patient products for screening or auxiliary screening SARS-CoV-2.
9. Use of a kit according to any one of claims 5 to 7, for any one of:
x1, detecting or assisting in detecting SARS-CoV-2;
x2, preparing or auxiliary detecting SARS-CoV-2 product;
x3, detecting or assisting to detect whether the sample to be detected contains SARS-CoV-2;
x4, preparing and detecting or detecting in an auxiliary way whether the sample to be detected contains SARS-CoV-2 products;
x5, detecting or assisting in detecting the D614G mutant strain of SARS-CoV-2;
x6, preparing D614G mutant strain product for detecting or assisting in detecting SARS-CoV-2;
x7, detecting or detecting in an auxiliary way whether the sample to be detected contains the D614G mutant strain of SARS-CoV-2;
x8, preparing a D614G mutant strain product for detecting or assisting in detecting whether the sample to be detected contains SARS-CoV-2;
x9, detecting or assisting to detect whether SARS-CoV-2 generates D614G mutation;
x10, preparing a product for detecting or assisting in detecting whether SARS-CoV-2 generates D614G mutation;
x11, diagnosis or auxiliary diagnosis of SARS-CoV-2 caused disease;
x12, preparing a product for diagnosing or assisting in diagnosing diseases caused by SARS-CoV-2;
x13, diagnosis or auxiliary diagnosis of diseases caused by D614G mutant strain of SARS-CoV-2;
x14, preparing a product for diagnosing or assisting in diagnosing diseases caused by the D614G mutant strain of SARS-CoV-2;
x15, screening or auxiliary screening of SARS-CoV-2 infected patients;
x16, preparing products for screening or auxiliary screening SARS-CoV-2 infection patients;
x17, screening or auxiliary screening for SARS-CoV-2D 614G mutant infected patients;
x18, preparing D614G mutant infected patient products for screening or auxiliary screening SARS-CoV-2.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202110900752.3A CN113604609B (en) | 2021-08-06 | 2021-08-06 | Primer combination for detecting SARS-CoV-2 and D614G mutant strain thereof and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202110900752.3A CN113604609B (en) | 2021-08-06 | 2021-08-06 | Primer combination for detecting SARS-CoV-2 and D614G mutant strain thereof and application thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN113604609A true CN113604609A (en) | 2021-11-05 |
| CN113604609B CN113604609B (en) | 2022-11-15 |
Family
ID=78307391
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202110900752.3A Active CN113604609B (en) | 2021-08-06 | 2021-08-06 | Primer combination for detecting SARS-CoV-2 and D614G mutant strain thereof and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN113604609B (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114686620A (en) * | 2022-01-21 | 2022-07-01 | 生物岛实验室 | Novel primer combination, kit and detection method for mass spectrometric detection of multiple variant strains of coronavirus |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111471804A (en) * | 2020-06-05 | 2020-07-31 | 浙江迪谱诊断技术有限公司 | Kit for detecting novel coronavirus with high sensitivity and high throughput and application thereof |
-
2021
- 2021-08-06 CN CN202110900752.3A patent/CN113604609B/en active Active
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111471804A (en) * | 2020-06-05 | 2020-07-31 | 浙江迪谱诊断技术有限公司 | Kit for detecting novel coronavirus with high sensitivity and high throughput and application thereof |
Non-Patent Citations (3)
| Title |
|---|
| ELHAM SHEIKHZADEH等: "diagnostic techniques for COVID-19 and new developments", 《TALANTA》 * |
| FEI ZHAO等: "A novel strategy for the detection of SARS-CoV-2 variants based on multiplex PCR-MALDI-TOF MS", 《MEDRXIV》 * |
| PETRA WANDERNOTH等: "Detection of severe acute respiratory syndrome coronavirus 2(SARS-CoV-2) by mass spectrometry", 《VIRUSES》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114686620A (en) * | 2022-01-21 | 2022-07-01 | 生物岛实验室 | Novel primer combination, kit and detection method for mass spectrometric detection of multiple variant strains of coronavirus |
| CN114686620B (en) * | 2022-01-21 | 2024-05-07 | 生物岛实验室 | Novel primer combination, kit and detection method for detecting nucleic acid mass spectrum of various variants of coronaviruses |
Also Published As
| Publication number | Publication date |
|---|---|
| CN113604609B (en) | 2022-11-15 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| WO2021174984A1 (en) | Rt-pcr detection method and kit for novel coronavirus | |
| CN111020064B (en) | Novel coronavirus ORF1ab gene nucleic acid detection kit | |
| WO2021196498A1 (en) | Primer, probe and kit for detecting novel coronavirus | |
| EP3882364B1 (en) | Dual detection kit for 2019 novel corona virus | |
| WO2022095723A1 (en) | Kit and method for detecting sars-cov-2 | |
| CN110760620A (en) | Classical swine fever virus and African classical swine fever virus dual-fluorescence PCR detection reagent, kit and detection method | |
| CN113774168A (en) | 2019 novel coronavirus, Deltay and lambda variant strain typing nucleic acid detection kit and detection method thereof | |
| CN110643745A (en) | Composition and kit for detecting feline calicivirus and feline parvovirus and application thereof | |
| CN111961761A (en) | Primer probe group and kit for detecting different genotypes of porcine circovirus | |
| CN114214458B (en) | Multiplex fluorescent quantitative PCR (polymerase chain reaction) primer and probe for simultaneously detecting four pig reproductive disorder pathogens and method thereof | |
| CN113604609B (en) | Primer combination for detecting SARS-CoV-2 and D614G mutant strain thereof and application thereof | |
| CN111471800B (en) | Kit for detecting novel coronavirus and amplification primer composition thereof | |
| CN116004920B (en) | Fluorescence PCR detection method and kit for four different lineages of strains of porcine reproductive and respiratory syndrome | |
| CN110592285A (en) | RAA primer probe for detecting Nipah virus and detection method | |
| CN113801963B (en) | Primer probe combination, kit and method for detecting coronavirus | |
| CN112626278B (en) | Primer and probe for identifying canine distemper virus wild strain and vaccine strain and application | |
| CN112779250B (en) | Kit for identifying porcine reproductive and respiratory syndrome virus and/or African swine fever virus and application thereof | |
| CN112266980A (en) | Novel coronavirus 2019-nCoV real-time fluorescent PCR detection primer, probe, kit and method | |
| CN112029901A (en) | Reagent for improving specificity of nucleic acid amplification reaction, nucleic acid amplification reaction solution and kit | |
| CN116814862B (en) | Identification and detection of African swine fever gene type I virulent virus, type I attenuated virus and type II virus | |
| CN111004868A (en) | Fluorescent PCR (polymerase chain reaction) primer, probe and kit for detecting goat intranasal tumor virus | |
| CN111206117A (en) | Kit for detecting human immunodeficiency virus | |
| CN114592090B (en) | Dual-fluorescence quantitative PCR detection primer, probe and kit for identifying genes I and II of African swine fever virus | |
| CN114262758B (en) | Kit for detecting novel coronavirus mutant strain and detection method | |
| CN115927743B (en) | Composition, kit and method for simultaneously detecting HSV-1, HSV-2 and VZV |
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 |