Novel 2019 coronavirus S protein gene isothermal chromogenic amplification primer group, screening kit and detection method

A coronavirus, amplification primer technology, applied in the biological field

Pending Publication Date: 2020-10-23
TIANJIN NORMAL UNIVERSITY +1
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  • Abstract
  • Description
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  • Application Information

AI Technical Summary

Problems solved by technology

Solve the problem that currently only real-time fluorescent quantitative RT-PCR method and kits are used to detect ORF1ab and N genes for nucleic acid detection of new coronavirus, and constant temperature amplification chip method is used to detect S gene and N gene, but there is no constant temperature chromogenic RT-PCR detection and screening method And the problem of kit screening and detection of S protein gene

Method used

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  • Novel 2019 coronavirus S protein gene isothermal chromogenic amplification primer group, screening kit and detection method
  • Novel 2019 coronavirus S protein gene isothermal chromogenic amplification primer group, screening kit and detection method
  • Novel 2019 coronavirus S protein gene isothermal chromogenic amplification primer group, screening kit and detection method

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0052] Example 1 S protein gene constant temperature chromogenic RT-PCR amplification primer set screening and specificity verification

[0053] 1.1 Design of primer set

[0054] 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 / ).

[0055] 1.2 Sequence alignment of specific amplified gene fragments

[0056] 1.2.1 Intraspecies conservative comparison

[0057] The 26 sets of 2...

Embodiment 2

[0082] The optimization of embodiment 2 S protein gene constant temperature chromogenic RT-PCR amplification detection system

[0083] 2.1 Establishment of constant temperature chromogenic RT-PCR amplification reaction system

[0084] Using 8U / L Bst enzyme, 5U / L AMV enzyme, 2.8mM dNTPs, 40mM Tris-HC (pH=8.3), 20mM KCl, 16mM MgSO 4 , 20mM (NH 4 ) 2 SO 4 , 1.6M betaine to prepare a constant temperature chromogenic amplification premixed reaction solution, using self-designed and confirmed the most specific fourth set of primers (inner primers, outer primers and loop primers) for the detection of the new coronavirus S protein gene to prepare a new The reaction system of constant temperature color development amplification detection of coronavirus S protein gene is mixed and placed in a constant temperature instrument for constant temperature color development RT-PCR amplification.

[0085] The reaction conditions are: (the corresponding reaction conditions can be set accordin...

Embodiment 3

[0092] Example 3 Sensitivity verification of S protein gene constant temperature chromogenic RT-PCR amplification screening detection method

[0093] 3.1 Sensitivity test method

[0094] 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, and 10copies / μL were detected by the optimized constant temperature chromogenic amplification reaction system to verify the constant temperature chromogenic amplification inner and outer loop primers of the new coronavirus S protein gene designed in this case The sensitivity of the screening assay established by the group.

[0095] 3.2 Sensitivity test results

[0096] According to the method of 3.1, the sensitivity test was carried out, and the results showed that the fourth set of primers was used to carry out the detection sensitivity test of the c...

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Abstract

The invention relates to a 2019 novel coronavirus S protein gene isothermal chromogenic amplification primer group, a screening kit and a detection method, and belongs to the technical field of biology. The novel coronavirus S protein gene isothermal chromogenic RT-PCR amplification inner and outer loop primer group, the isothermal chromogenic screening detection method and the screening kit whichare developed by the invention solve the problems that: only an isothermal amplification chip method is used for detecting the S protein gene, and isothermal chromogenic screening methods and kits donot use cheap and convenient isothermal water bath kettle and metal bath for detecting the S protein gene. The invention is a powerful supplement to a novel coronavirus S protein gene detection method and kit. Compared with similar technologies at home and abroad, the kit and method have obvious cost and benefit advantages. According to the developed novel coronavirus S protein gene chromogenic chromogenic RT-PCR screening detection kit, the performance index of the kit is the same as that of fluorescent quantitative RT-PCR, the detection time is short, and the operation is simple, convenientand rapid.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a primer set, a screening kit and a detection method for the 2019 novel coronavirus S protein gene constant temperature color development amplification. 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 environm...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/6844C12N15/11
CPCC12Q1/701C12Q1/6844C12Q2600/166C12Q2531/119C12Q2537/1376C12Q2521/107C12Q2545/113Y02A50/30
Inventor 郑文杰曹际娟郑秋月季超
Owner TIANJIN NORMAL UNIVERSITY
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