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Isothermal fluorescent amplification primer group for S protein gene of 2019-nCoV, kit and detection method

A fluorescent kit, coronavirus technology, applied in the biological field, can solve the problem of lack of S protein gene, etc., and achieve the effect of low testing cost

Pending Publication Date: 2020-10-02
DALIAN NATIONALITIES UNIVERSITY +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Solve the problem that currently there are only real-time fluorescent quantitative RT-PCR methods and kits for detection of ORF1ab and N genes for the detection of new coronavirus nucleic acids, and the co

Method used

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  • Isothermal fluorescent amplification primer group for S protein gene of 2019-nCoV, kit and detection method
  • Isothermal fluorescent amplification primer group for S protein gene of 2019-nCoV, kit and detection method
  • Isothermal fluorescent amplification primer group for S protein gene of 2019-nCoV, kit and detection method

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0053] Example 1 Primer set screening and specificity verification for constant temperature fluorescent RT-PCR detection of S protein gene

[0054] 1.1 Design of primer sets

[0055]According to the novel coronavirus national science and technology resource service system (http: / / nmdc.cn / # / nCoV), 26 groups of 2019-nCoV sequences have been downloaded and uploaded, the conserved region of the S protein gene is selected, and 6 groups are designed by Primer Premier 5.0 software The constant temperature fluorescent amplification primer pairs are respectively marked as the first group, the second group, the third group, the fourth group, the fifth group and the sixth group. Each set of primers includes 2 inner primers, 2 outer primers and 2 loop primers, and was evaluated using Primer blast (http: / / www.ncbi.nlm.nih.gov / tools / primer-blast / ).

[0056] 1.2 Sequence alignment of specific amplified gene fragments

[0057] 1.2.1 Intraspecies conservative comparison

[0058] The 26 sets...

Embodiment 2

[0083] The optimization of embodiment 2S protein gene constant temperature fluorescent RT-PCR detection system

[0084] 2.1 Establishment of constant temperature fluorescent RT-PCR reaction system

[0085] The 25 μL reaction system includes 2.0 μL of RNA template, 12.5 μL of reaction solution RM-SCI (Guangzhou Double Helix Gene Technology Co., Ltd., China, containing 2.8mM dNTPs, 40mM Tris-HCl, 20mM KCl, 16mM MgSO 4 , 20mM (NH 4 ) 2 SO 4 , 1.6M betaine), 0.5 μL of 1000×SYBR Green I (Thermo Scientific), 0.8 μL of 8U / μL Bst polymerase (NEB), 0.2 μL of 5U / μL AMV enzyme (TAKARA, China, Dalian), 8 μL of Ultrapure water, 1.0 μL of inner primer, outer primer and loop primer. Finally, add 1 drop (about 20 μL) of paraffin to seal the tube. Prepare a constant temperature fluorescent RT-PCR detection reaction system for the new coronavirus S protein gene, mix it and place it in a GENIE isothermal amplification instrument or real-time fluorescent quantitative PCR instrument for const...

Embodiment 3

[0094] Example 3S protein gene constant temperature fluorescent RT-PCR detection method sensitivity verification

[0095] 3.1 Sensitivity test method

[0096] Dilute the positive control samples of the S protein gene in vitro transcription RNA plasmid with a 10-fold difference, and the gradient concentrations are 1×10^6 copies / μL, 1×10^5 copies / μL, 1×10^4 copies / μL, 1×10 ^3copies / μL, 1×10^2copies / μL, 10copies / μL were detected by the optimized fluorescent constant temperature RT-PCR reaction system to verify the inside and outside of the constant temperature fluorescent RT-PCR detection of the new coronavirus S protein gene designed in this case The sensitivity of the isothermal fluorescent RT-PCR detection method established by the loop primer set.

[0097] 3.2 Sensitivity test results

[0098] The sensitivity test was carried out according to the method in 3.1, and the results showed that RNA at a concentration of 0.1fg / μL (10copies / μL) was probabilistically detected. Thre...

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Abstract

The invention relates to an isothermal fluorescent amplification primer group for an S protein gene of 2019-nCoV, a kit and a detection method, and belongs to the technical field of biology. A whole genome sequence of an internationally reported novel coronavirus are closely analyzed, intraspecific conservation comparison, extraspecific homology comparison and difference site analysis are carriedout on the S protein gene of the novel coronavirus, and two inner primers, two outer primers and two loop primers are determined as a core technique through designing and screening, so that the guarantee of the specificities and sensitivities of an isothermal fluorescent RT-PCR detection method and the kit is enhanced. Compared with domestic and overseas similar techniques, a result of the invention has the obvious advantages on the cost and the benefit. The researched and developed isothermal fluorescent RT-PCR detection kit for the S protein gene of novel coronavirus has the performance indexes equal to fluorescent quantitative RT-PCR, the detection time can be 30-45 minutes, and the operation is easy, convenient and rapid.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a primer set, a kit and a detection method for constant temperature fluorescence amplification of the 2019 novel coronavirus S protein gene. Background technique [0002] At the beginning of 2020, a sudden new coronavirus epidemic has brought severe challenges to people's life, health and production. The key to epidemic prevention and control lies in early detection, early isolation, early diagnosis, and early treatment. Major medical institutions urgently need a clear diagnosis to fight the epidemic. Among them, nucleic acid detection methods and reagents play the most important role in the diagnosis and are the gold standard for epidemic diagnosis. However, at present, nucleic acid detection reagents are not only in short supply, but also have the problem of low detection rate and frequent false negatives; and due to the strict requirements of PCR experimental environment and faci...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/686C12N15/11C12R1/93
CPCC12Q1/701C12Q1/686C12Q2521/107C12Q2521/101C12Q2563/107
Inventor 曹际娟郑文杰郑秋月季超
Owner DALIAN NATIONALITIES UNIVERSITY
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