CN113604609B - Primer combination for detecting SARS-CoV-2 and D614G mutant strain thereof and application thereof - Google Patents
Primer combination for detecting SARS-CoV-2 and D614G mutant strain thereof and application thereof Download PDFInfo
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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 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 thereof and application thereof in the field of biomedicine.
Background
Coronaviruses (HCoV) mainly cause respiratory tract and gastrointestinal tract infections, and are 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, severe acute respiratory syndrome coronavirus (SARS-CoV) and middle east respiratory syndrome coronavirus (MERS-CoV) of the genus betacoronavirus. 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.
Since 12 months in 2019, the novel coronavirus SARS-CoV-2 rapidly spreads in large area around the world and gradually becomes variant, so that the infectivity is strong and the harm is serious. 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 accounts for less than 10% of the global neocoronavirus sequencing sequence 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 for detecting, identifying and typing 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 for coding 614 th aspartic acid in an S gene is mutated from GAT to a codon GGT for glycine.
In order to solve the technical problem, the invention firstly provides a primer combination (marked as primer combination 1), wherein the primer combination 1 consists of primer pairs with the names of ORF1b-P, N1-P, N2-P, S-P and S (D614G) -P respectively and MPE primers with the names of ORF1b-T, N1-T, N2-T, S-T and S (D614G) -T respectively;
the ORFs 1b-P are composed of single-stranded DNAs with the names of ORF1b-F and ORF1b-R respectively, and the sequences of the ORFs 1b-F and the ORFs 1b-R are sequences 1 and 2 respectively;
the N1-P consists of single-stranded DNA with the names of N1-F and N1-R respectively, and the sequences of the N1-F and the N1-R are respectively sequences 4 and 5;
the N2-P consists of single-stranded DNA with the names of N2-F and N2-R respectively, and the sequences of the N2-F and the N2-R are respectively sequences 7 and 8;
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 with the names S (D614G) -F and S (D614G) -R, respectively, and 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 single-stranded DNA shown as a sequence 6 in a sequence table;
the N2-T is a single-stranded DNA shown as a sequence 9 in a sequence table;
the S-T is a 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 a sequence table.
In the primer set 1, the number of moles of the ORFs 1b to F, the ORFs 1b to R, the N1 to F, the N1 to R, the N2 to F, the N2 to R, the S-F, the S-R, the S (D614G) -F, and the S (D614G) -R may be equal to each other.
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 to detect SARS-CoV-2;
x2, preparing a product for detecting or assisting in detecting SARS-CoV-2;
x3, detecting or assisting to detect whether the sample to be detected contains SARS-CoV-2;
x4, preparing a sample to be detected or detecting whether the sample to be detected contains SARS-CoV-2 products in an auxiliary way;
x5, detecting or assisting to detect a D614G mutant strain of SARS-CoV-2;
x6, preparing a 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 a D614G mutant strain of SARS-CoV-2;
x8, preparing and detecting or assisting to detect whether the sample to be detected contains a D614G mutant strain product of SARS-CoV-2;
x9, detecting or assisting to detect whether the SARS-CoV-2 generates D614G mutation;
x10, preparing a product for detecting or assisting in detecting whether the 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 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 a product for screening or assisting in screening SARS-CoV-2 infected patients;
x17, screening or auxiliary screening of a patient infected by the D614G mutant strain of SARS-CoV-2;
x18, preparing a product for screening or assisting in screening SARS-CoV-2D 614G mutant infected patients.
The invention also provides another primer combination (marked as a primer combination 2), wherein the primer combination 2 consists 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 with the names S (D614G) -F and S (D614G) -R, respectively, and 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 a 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, detecting or detecting the D614G mutant strain of SARS-CoV-2 in an auxiliary way;
y2, preparing a D614G mutant strain product for detecting or assisting in detecting SARS-CoV-2;
y3, detecting or detecting in an auxiliary way whether the sample to be detected contains the D614G mutant strain of SARS-CoV-2;
y4, preparing a D614G mutant strain product for detecting whether the sample to be detected contains SARS-CoV-2 or not in an auxiliary way;
y5, detecting or assisting to detect whether the 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 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 of a patient infected by the D614G mutant strain of SARS-CoV-2;
y10, preparing products for screening or assisting in screening D614G mutant strain infected patients of 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-SJ 09-SNPs) (thawing Biotechnology (Qingdao) Ltd.), such as A1 component: PCR premix, SAP enzyme, SAP buffer, MPE enzyme, MPE buffer, E-ddNTPmix, matrix fluid, resin, oligoplate.
The kit may have any one of the following uses:
x1, detecting or auxiliary detecting SARS-CoV-2;
x2, preparing a product for detecting or assisting in detecting SARS-CoV-2;
x3, detecting or assisting to detect whether the sample to be detected contains SARS-CoV-2;
x4, preparing and detecting or assisting to detect 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 a 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 a D614G mutant strain of SARS-CoV-2;
x8, preparing and detecting or assisting to detect whether the sample to be detected contains the D614G mutant strain product of SARS-CoV-2;
x9, detecting or assisting to detect whether the SARS-CoV-2 generates D614G mutation;
x10, preparing a product for detecting or assisting in detecting whether the 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 a disease caused by the D614G mutant 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 of SARS-CoV-2 infected patients;
x17, screening or auxiliary screening of a patient infected by the D614G mutant strain of SARS-CoV-2;
and X18, preparing a product for screening or assisting in screening the D614G mutant strain infected patient of 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 to detect SARS-CoV-2;
x2, preparing a product for detecting or assisting in detecting SARS-CoV-2;
x3, detecting or assisting to detect whether the sample to be detected contains SARS-CoV-2;
x4, preparing and detecting or assisting to detect 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 a 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 and detecting or assisting to detect whether the sample to be detected contains a D614G mutant strain product of SARS-CoV-2;
x9, detecting or assisting to detect whether the SARS-CoV-2 generates D614G mutation;
x10, preparing a product for detecting or assisting to detect whether the 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 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 of SARS-CoV-2 infected patients;
x17, screening or auxiliary screening of a patient infected by the D614G mutant strain of SARS-CoV-2;
x18, preparing a product for screening or assisting in screening SARS-CoV-2D 614G mutant infected patients.
The following applications of the kit also belong to the protection scope of the invention:
x1, detecting or auxiliary detecting SARS-CoV-2;
x2, preparing a product for detecting or assisting in detecting SARS-CoV-2;
x3, detecting or assisting to detect whether the sample to be detected contains SARS-CoV-2;
x4, preparing and detecting or assisting to detect whether the sample to be detected contains SARS-CoV-2 products;
x5, detecting or assisting to detect a D614G mutant strain of SARS-CoV-2;
x6, preparing a 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 a D614G mutant strain of SARS-CoV-2;
x8, preparing and detecting or assisting to detect whether the sample to be detected contains a D614G mutant strain product of SARS-CoV-2;
x9, detecting or assisting to detect whether the SARS-CoV-2 generates D614G mutation;
x10, preparing a product for detecting or assisting in detecting whether the 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 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 a product for screening or assisting in screening SARS-CoV-2 infected patients;
x17, screening or auxiliary screening of a patient infected by the D614G mutant strain of SARS-CoV-2;
and X18, preparing a product for screening or assisting in screening the D614G mutant strain infected patient of 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, and no cross reaction occurs with other 6 kinds of coronavirus infecting human, thus the false negative result can be obviously reduced. The method has the advantages of 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. In the quantitative tests in the following examples, three replicates were set up and the results averaged. In the following examples, 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, unless otherwise specified.
SNP typing kit (QT-SJ 09-SNPs) Intelligent bioscience (Qingdao) Co., ltd, which contains the following reagents:
example 1 preparation of primer combination for detection of novel coronavirus
1. Preparation of primer combinations
This example prepares a primer set for detecting a novel coronavirus, which consists of a primer pair designated ORF1b-P, N1-P, N2-P, S-P and S (D614G) -P, respectively, and MPE primers designated ORF1b-T, N1-T, N2-T, S-T and S (D614G) -T, respectively. ORF1b-P consists of single-stranded DNA with the names ORF1b-F and ORF1b-R, respectively, and the sequences are sequence 1 and sequence 2, respectively; N1-P is composed of single-stranded DNA with the names of N1-F and N1-R respectively, and the sequences of the single-stranded DNA are respectively sequence 4 and sequence 5; N2-P is composed of single-stranded DNA with the names of N2-F and N2-R respectively, and the sequences of the single-stranded DNA are respectively 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 consists of single-stranded DNA with the names S (D614G) -F and S (D614G) -R, respectively, and the sequences are respectively 13 and 14; the sequences of ORFs 1b-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 and S (D614G) -R are equal;
the molar ratio of ORFs 1b-T, N1-T, N2-T, S-T and S (D614G) -T was 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 two plasmids, pUC57-HCoV-HKU1-1 and pUC57-HCoV-HKU1-2, were selectively synthesized for HCoV-HKU1 due to the relatively large number of mutation sites of the intraspecies conserved sequences.
Construction of recombinant plasmid pUC57-SARS-CoV-2 and pUC57-SARS-CoV-2 containing the DNA fragment of SARS-CoV-2 the DNA fragment between the SacI and SalI recognition sequences of the pUC57 plasmid was replaced with the DNA fragment shown in sequence 16 in the sequence listing to obtain a recombinant plasmid.
Construction of recombinant plasmids pUC57-HCoV-229E and pUC57-HCoV-229E containing the DNA fragment of HCoV-229E are recombinant plasmids obtained 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.
Constructing a recombinant plasmid pUC57-HCoV-OC43 containing the DNA fragment of HCoV-OC43, wherein pUC57-HCoV-OC43 is obtained by replacing the DNA fragment between XbaI and SalI recognition sequences of pUC57 plasmid with the DNA fragment shown in sequence 18 in the sequence table.
A recombinant plasmid pUC57-HCoV-NL63 containing the DNA fragment of HCoV-NL63 was constructed, and pUC57-HCoV-NL63 was obtained by replacing the DNA fragment between BamHI and SalI recognition sequences of pUC57 plasmid with the DNA fragment shown in sequence 19 of the sequence Listing.
Construction of pUC57-HCoV-HKU1-1, pUC57-HCoV-HKU1-1 was a recombinant plasmid 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-HKU1-2, pUC57-HCoV-HKU1-2 was 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 by sequence 21 in the sequence listing.
A recombinant plasmid pUC57-SARS-CoV containing a DNA fragment of SARS-CoV was constructed, and pUC57-SARS-CoV was obtained by replacing the DNA fragment between the BamHI and SalI recognition sequences of the pUC57 plasmid with the DNA fragment shown in sequence No. 22 in the sequence Listing.
Constructing a recombinant plasmid containing a DNA fragment of MERS-CoV, wherein pUC57-MERS-CoV is the recombinant plasmid obtained by replacing the DNA fragment between the BamHI recognition sequence and the SalI recognition sequence 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-SJ 09-SNPs) by the following steps:
1. preparation of primer mixture
Preparing a primer mixed solution: the 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) 2 copies/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 ℃; 1, 10 cycles of 95 ℃, 15, 59 ℃, 30s,72 ℃ and 30s; 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)
Reaction system to the SAP digest product of step 3 the following components were added:
| 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 μ M, 8.72 μ M, 11.09 μ M, 9.99 μ M and 11.99 μ M. The obtained reaction system is reacted under the following conditions to obtain a mass extension product:
5. resin desalination
(1) After the reaction of step 4, 14. Mu.l of RNase-free ddH2O was added to each reaction well.
(2) The eight rows of tubes containing resin were gently inverted and snapped onto the sample plate to ensure that the resin holes were aligned with each hole in the sample. The resin tube was then tapped to drop the resin into the wells of the sample plate.
(3) And a palm centrifuge is used for instantaneous centrifugation to avoid the resin from being attached to the pipe wall.
(4) The sample plate with the resin was placed in an inverted mixer and mixed for 30min at 20 rpm.
(5) After the mixing is finished, the sample is centrifuged at 2000rpm for 1min, and the supernatant is collected to be tested.
6. Nucleic acid mass spectrometry to be detected
(1) Mixing the matrix solution, and performing ultrasonic treatment for 2min. And (3) putting 1 mu l of matrix at the center of the target spot, standing at room temperature for 4-5min, and naturally drying.
(2) Sucking 0.5 mul of supernatant obtained in the step 5 on the substrate, drying and crystallizing to be tested.
(3) And (4) 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 installation:
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 a set of calibrators (4 k-10kDa, gloiopeltis Biotech (Qingdao) Inc.) of 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 based on 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 the N1, N2, ORF1b and S genes 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-HKU1-2, pUC57-SARS-CoV and pUC57-MERS-CoV of example 1 were each subjected to RNase-free ddH 2 O dissolution to obtain the plasmid with the concentration of 10 5 The 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-HKU1-2 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 from 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, respectively 1 Copy/. Mu.l, 10 2 Copy/. Mu.l, 10 3 Copy/. Mu.l, 10 4 Copy/. Mu.l, 10 5 Copies/. Mu.l plasmid dilution.
The sensitivity of the primer set 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 5 amplification of different concentrations of pUC57-SARS-CoV-2 at 45 cycles
| Plasmid concentration (copies/. Mu.L) -repeats | N1 | N2 | ORF1b | S | S(D614G) |
| 10 0 Repetition of 1 | No amplification | Without amplification | No amplification | Without amplification | No amplification |
| 10 0 Repetition of 2 | Without amplification | No amplification | Without amplification | No amplification | Without amplification |
| 10 1 Repetition of 1 | G | G | A | G | A |
| 10 1 Repetition 2 | G | G | A | G | A |
| 10 2 Repetition of 1 | G | G | A | G | A |
| 10 2 Repetition of 2 | G | G | A | G | A |
| 10 3 Repetition of 1 | G | G | A | G | A |
| 10 3 Repetition of 2 | G | G | A | G | A |
| 10 4 Repetition of 1 | G | G | A | G | A |
| 10 4 Repetition of 2 | G | G | A | G | A |
| 10 5 Repetition of 1 | G | G | A | G | A |
| 10 5 Repetition 2 | G | G | A | G | A |
Example 5 and application of primer combination of example 1 to detection of actual sample
Throat 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). Of the 3 positives, 2 were further typed as D614G mutants (numbered 9-10).
The specific procedures for detecting clinical samples using the primer combination 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 positive for all 4 target genes N1, N2, ORF1b, S and S (D614G) of SARS-CoV-2 with MPE and a signal-to-noise ratio of > 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 the positive sample was D614G mutant.
As is clear from Table 15, the sample No. 10 was positive for the occurrence of MPE in all of the 3 target genes N1, S and S (D614G) of SARS-CoV-2.
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, the sample No. 11 was positive as it was confirmed that MPE was generated in all of the N1, N2, and ORF1b 3 target genes of SARS-CoV-2.
As is clear from Table 16, the fact that no MPE occurred in the S gene (containing the D614G mutation site) in sample No. 11 indicates that SARS-CoV-2 in this positive sample is not a 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 6 clinical sample test results
Table 7 clinical sample test results
TABLE 8 clinical specimen test results
TABLE 9 clinical specimen test results
TABLE 10 clinical specimen test results
Table 11 clinical sample test results
TABLE 12 clinical specimen test results
Table 13 clinical sample test results
TABLE 14 clinical specimen test results
Table 15 clinical sample test results
TABLE 16 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 the people's liberation army of China
<120> primer combination for detecting SARS-CoV-2 and D614G mutant strain thereof 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
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<212> DNA
<213> Artificial sequence (Artificial sequence)
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gcctgaccag taccagtgtg 20
<210> 3
<211> 26
<212> DNA
<213> Artificial sequence (Artificial sequence)
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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
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<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 (7)
1. The primer combination consists of five pairs of primer pairs with the names of ORF1b-P, N1-P, N2-P, S-P and S (D614G) -P respectively and five MPE primers with the names of ORF1b-T, N1-T, N2-T, S-T and S (D614G) -T respectively;
the ORFs 1b-P are composed of single-stranded DNAs with the names of ORF1b-F and ORF1b-R respectively, and the sequences of the ORFs 1b-F and the ORFs 1b-R are sequence 1 and sequence 2 respectively;
the N1-P consists of single-stranded DNA with the names of N1-F and N1-R respectively, and the sequences of the N1-F and the N1-R are respectively sequence 4 and sequence 5;
the N2-P consists of single-stranded DNA with the names of N2-F and N2-R respectively, and the sequences of the N2-F and the N2-R are respectively a sequence 7 and a sequence 8;
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 a sequence 10 and a sequence 11 respectively;
the S (D614G) -P consists of single-stranded DNA with the names S (D614G) -F and S (D614G) -R, and the sequences of the S (D614G) -F and the S (D614G) -R are sequence 13 and sequence 14 respectively;
the ORF1b-T is a single-stranded DNA with the sequence shown as a sequence 3;
the N1-T is single-stranded DNA with a sequence shown as a sequence 6;
the N2-T is single-stranded DNA with a sequence shown as a sequence 9;
the S-T is single-stranded DNA with a sequence shown as a sequence 12;
the S (D614G) -T is single-stranded DNA with the sequence shown as the sequence 15.
2. The primer combination of claim 1, wherein: the primer combination has any one of the following purposes:
x1, detecting or auxiliary detecting SARS-CoV-2;
x2, preparing a product for detecting or assisting in detecting SARS-CoV-2;
x3, detecting or assisting to detect a D614G mutant strain of SARS-CoV-2;
x4, preparing a product for detecting or assisting in detecting the D614G mutant strain of SARS-CoV-2;
x5, detecting or assisting to detect whether the SARS-CoV-2 generates D614G mutation;
x6, preparing a product for detecting or assisting in detecting whether the SARS-CoV-2 has D614G mutation;
x7, diagnosis or auxiliary diagnosis of SARS-CoV-2 caused disease;
x8, preparing a product for diagnosing or assisting in diagnosing diseases caused by SARS-CoV-2;
x9, diagnosis or auxiliary diagnosis of a disease caused by the D614G mutant of SARS-CoV-2;
x10, preparing a product for diagnosing or assisting in diagnosing diseases caused by the D614G mutant strain of SARS-CoV-2;
x11, screening or auxiliary screening of SARS-CoV-2 infected patients;
x12, preparing a product for screening or auxiliary screening of SARS-CoV-2 infected patients;
x13, screening or auxiliary screening of a patient infected by the D614G mutant strain of SARS-CoV-2;
x14, preparing products for screening or auxiliary screening of SARS-CoV-2D 614G mutant infected patients.
3. A kit comprising the primer combination of claim 1.
4. The kit of claim 3, wherein: the kit also contains reagents required for performing PCR amplification and/or reagents required for mass extension.
5. The kit according to claim 3 or 4, characterized in that: the kit has any one of the following uses:
x1, detecting or auxiliary detecting SARS-CoV-2;
x2, preparing a product for detecting or assisting in detecting SARS-CoV-2;
x3, detecting or assisting to detect a D614G mutant strain of SARS-CoV-2;
x4, preparing a product for detecting or assisting in detecting the D614G mutant strain of SARS-CoV-2;
x5, detecting or assisting to detect whether the SARS-CoV-2 generates D614G mutation;
x6, preparing a product for detecting or assisting in detecting whether the SARS-CoV-2 has D614G mutation;
x7, diagnosis or auxiliary diagnosis of SARS-CoV-2 caused disease;
x8, preparing a product for diagnosing or assisting in diagnosing diseases caused by SARS-CoV-2;
x9, diagnosis or auxiliary diagnosis of diseases caused by D614G mutant strain of SARS-CoV-2;
x10, preparing a product for diagnosing or assisting in diagnosing diseases caused by the D614G mutant strain of SARS-CoV-2;
x11, screening or auxiliary screening of SARS-CoV-2 infected patients;
x12, preparing a product for screening or auxiliary screening of SARS-CoV-2 infected patients;
x13, screening or auxiliary screening of a patient infected by the D614G mutant strain of SARS-CoV-2;
and X14, preparing products for screening or assisting in screening the D614G mutant strain infected patients of SARS-CoV-2.
6. Use of a primer combination according to claim 1 or 2 for any one of the following applications:
x1, preparing a product for detecting or assisting in detecting SARS-CoV-2;
x2, preparing a product for detecting or assisting in detecting the D614G mutant strain of SARS-CoV-2;
x3, preparing a product for detecting or assisting in detecting whether the SARS-CoV-2 has D614G mutation;
x4, preparing a product for diagnosing or assisting in diagnosing diseases caused by SARS-CoV-2;
x5, preparing a product for diagnosing or assisting in diagnosing diseases caused by the D614G mutant strain of SARS-CoV-2;
x6, preparing a product for screening or auxiliary screening of SARS-CoV-2 infected patients;
and X7, preparing a product for screening or assisting in screening the D614G mutant strain infected patient of SARS-CoV-2.
7. Use of a kit according to any one of claims 3 to 5 for any one of the following:
x1, preparing a product for detecting or assisting in detecting SARS-CoV-2;
x2, preparing a product for detecting or assisting in detecting the D614G mutant strain of SARS-CoV-2;
x3, preparing a product for detecting or assisting in detecting whether the SARS-CoV-2 has D614G mutation;
x4, preparing a product for diagnosing or assisting in diagnosing diseases caused by SARS-CoV-2;
x5, preparing a product for diagnosing or assisting in diagnosing diseases caused by the D614G mutant strain of SARS-CoV-2;
x6, preparing a product for screening or auxiliary screening of SARS-CoV-2 infected patients;
and X7, preparing a product for screening or assisting in screening the D614G mutant strain infected patient of SARS-CoV-2.
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