Primer combination for detecting SARS-CoV-2 and D614G mutant strain thereof and application of primer combination

A technology of D614G and primer combination, applied in the field of biomedicine, can solve the problems of unfavorable case screening, high sample requirements, limited sample capacity, etc., and achieve the effect of reducing false negative results, good application prospects, and low detection cost

Active Publication Date: 2021-11-05
ACADEMY OF MILITARY MEDICAL SCI
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  • Description
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AI Technical Summary

Problems solved by technology

At present, the PCR detection kits for detecting SARS-CoV-2 have a large number of false negatives and a large number of positive results in the actual application process, and the daily processing capacity of samples is limited, which is not conducive to rapid, accurate and large-scale clinical case screening The requirements for samples are relatively high, and the clinical discharge standard requires three nucleic acid tests to be negative; similar to other coronavirus sequences that infect humans, the selection of target genes and specific sequences did not refer to other coronavirus sequences for specificity The sensitivity and accuracy of screening and primer probe detection need to be improved; only cases can be confirmed, and SARS-CoV-2 infected by confirmed cases cannot be further typed

Method used

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  • Primer combination for detecting SARS-CoV-2 and D614G mutant strain thereof and application of primer combination
  • Primer combination for detecting SARS-CoV-2 and D614G mutant strain thereof and application of primer combination
  • Primer combination for detecting SARS-CoV-2 and D614G mutant strain thereof and application of primer combination

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0119] Embodiment 1, the preparation of the primer combination that detects novel coronavirus

[0120] 1. Preparation of primer combinations

[0121] In this example, a primer combination for the detection of novel coronavirus was prepared. MPE primer composition for T, N1-T, N2-T, S-T and S(D614G)-T. ORF1b-P is composed of single-stranded DNA named ORF1b-F and ORF1b-R, and its sequences are sequence 1 and 2, respectively; N1-P is composed of single-stranded DNA named N1-F and N1-R, respectively. The sequences are sequence 4 and 5 respectively; N2-P is composed of single-stranded DNA named N2-F and N2-R respectively, and its sequence is sequence 7 and 8 respectively; S-P is composed of single-stranded DNA named S-F and S-R respectively , whose sequences are sequences 10 and 11, respectively; S(D614G)-P is composed of single-stranded DNA named S(D614G)-F and S(D614G)-R, respectively, and whose sequences are sequences 13 and 14, respectively; ORF1b- The sequences of T, N1-T, N2...

Embodiment 2

[0141] Embodiment 2. Establishment of a method for detecting novel coronaviruses using the primer combination of Embodiment 1

[0142] Detect the amplification efficiency of the primer combination of embodiment 1, adopt SNP typing kit (QT-SJ09-SNPs) to carry out, the steps are as follows:

[0143] 1. Prepare the primer mixture

[0144] Preparation of primer mixture: use RNase-free deionized water to dissolve the primer pairs ORF1b-P, N1-P, N2-P, S-P and S(D614G)-P in Example 1 to obtain a primer mixture. In the primer mixture , 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 all 0.5 μM.

[0145] 2. Multiplex PCR amplification

[0146] Use the primer mixture in step 1 to carry out multiplex PCR amplification, the DNA template is pUC57-SARS-CoV-2 in Example 1, and the system is as follows:

[0147] components volume / μl PCR master mix 2 Deionized water 1 primer mix 1 DNA template (at a co...

Embodiment 3

[0186] The specificity of the primer combination of embodiment 3, embodiment 1

[0187] pUC57-SARS-CoV-2, pUC57-HCoV-229E, pUC57-HCoV-OC43, pUC57-HCoV-NL63, pUC57-HCoV-HKU1-1, pUC57-HCoV-HKU1-2, pUC57-SARS of Example 1 –CoV and pUC57-MERS-CoV utilize RNase-free ddH respectively 2 O was dissolved, and the concentration of the plasmid obtained was 10 5 Eight plasmid solutions in copies / μl, namely 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.

[0188] Using the method 2-6 in Example 2, the DNA templates were respectively replaced with the above-mentioned eight kinds of plasmid solutions, and the other steps were kept unchanged, and the specificity of the primer combination in Example 1 was tested.

[0189] The results are shown in Table 4. The specificity of the primer combination in Example 1 is very good, th...

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Abstract

The invention discloses a primer combination for detecting SARS-CoV-2 and a D614G mutant strain thereof and application of the primer combination. The primer combination for detecting the SARS-CoV-2 and the D614G mutant strain thereof disclosed by the invention is composed of 15 single-stranded DNAs shown in sequences 1-15 in a sequence table. When the primer combination is used for detecting the novel coronavirus, the specificity is high, the sensitivity is high, the sample detection limit is 1-10 copy number / reaction, cross reaction with other six kinds of coronavirus infecting people is avoided, and false negative results can be remarkably reduced. The primer combination provided by the invention can be used for large-scale, rapid, in-vitro and qualitative detection of novel coronavirus genes in nasopharyngeal swab, sputum, alveolar lavage fluid and other samples of novel coronavirus infected pneumonia suspected cases, suspected aggregation case patients, and other diagnosers needing novel coronavirus infection diagnosis or identification, and has good application prospects.

Description

technical field [0001] The invention relates to a combination of primers for detecting SARS-CoV-2 and its D614G mutant strain and its application in the field of biomedicine. Background technique [0002] Coronaviruses (HCoVs) mainly cause respiratory and gastrointestinal infections and are genetically divided into four main genera: alphacoronaviruses, betacoronaviruses, gammacoronaviruses, and deltacoronaviruses. At present, six coronaviruses that infect humans have been identified, including HCoV-NL63 and HCoV-229E of the alphacoronavirus genus, HCoV-OC43, HCoV-HKU1, and severe acute respiratory syndrome coronavirus (SARS-CoV) of the betacoronavirus genus. and Middle East respiratory syndrome coronavirus (MERS-CoV). On February 11, 2020, the World Health Organization (WHO) named the 2019 novel coronavirus as COVID-19, and the International Committee on Taxonomy of Viruses (ICTV) named it severe acute respiratory syndrome coronavirus 2 (SARS-Cov-2) . SARS-CoV-2 is the se...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/686C12N15/11
CPCC12Q1/701C12Q1/686C12Q2537/143Y02A50/30
Inventor 辛文文康琳柳婷婷王菁王景林高姗李岩伟袁媛
Owner ACADEMY OF MILITARY MEDICAL SCI
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