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Primer probe set for detecting novel corona virus SARS-CoV-2 and detection method

A primer-probe, coronavirus technology, used in biochemical equipment and methods, DNA/RNA fragments, recombinant DNA technology, etc., can solve the problem of sample backlog of suspected cases, inability to obtain results quickly, and detection application limitations, etc. Short detection time, overcoming long detection time and good repeatability

Pending Publication Date: 2020-05-29
JIANGSU QITIAN GENE BIOTECHNOLOGY CO LTD +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the RT-PCR method requires expensive and complex instruments and equipment due to its variable temperature characteristics and requires professional and technical personnel to operate. The existing RT-PCR detection method for the new coronavirus SARS-CoV-2 requires on-site preparation of the reaction system. The reaction program is also divided into two steps. The first step is reverse transcription at 50-55°C for more than 30 minutes and amplification detection time for 40 to 90 minutes. The time required for the overall nucleic acid amplification detection depends on different kits. -3 hours, so the application of testing at the grassroots level and on-site is greatly restricted. Due to the limitation of testing speed and testing personnel, it will lead to a backlog of samples of a large number of suspected cases, and the results cannot be obtained quickly

Method used

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  • Primer probe set for detecting novel corona virus SARS-CoV-2 and detection method
  • Primer probe set for detecting novel corona virus SARS-CoV-2 and detection method
  • Primer probe set for detecting novel corona virus SARS-CoV-2 and detection method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0054] The primary screening of embodiment 1 specific primer probe combination

[0055] The full sequence of the new coronavirus SARS-CoV-2 announced by the Chinese Center for Disease Control and Prevention contains 10 genes for comprehensive analysis and comparison, combined with the needs of epidemic prevention and control, to achieve the purpose of rapid screening, the core solution of the present invention is fast, sensitive, Accurate these three basic points, select the most conservative ORF 1ab gene sequence from 10 genes to design primer probes, and in order to detect the new coronavirus SARS-CoV-2 faster and more accurately, targeting the ORF 1ab gene Perform single gene detection, reduce the interference of detection of multiple genes, improve the amplification speed and detection sensitivity;

[0056] The specific design uses DNAMAN software for coronavirus homology analysis and b1ast sequence analysis, and screens out two intervals with excellent conservation and sp...

Embodiment 2

[0075] The determination of embodiment 2 specific primer probe combinations

[0076] On the basis of the 5 primer probe combinations in the above-mentioned embodiment 1, further adaptive screening is carried out with the nucleic acid of the new coronavirus SARS-CoV-2 strain culture, and the selection of a pair of primer probes with the highest sensitivity is provided by the present invention. The primer probe set;

[0077] The purpose of this embodiment is to screen out the most sensitive primer probe group, and the nucleic acid stock solution is diluted to 10 times by 10 times. 2 、10 3 、10 4 、10 5 、10 6 、10 7 、10 8 、10 9 、10 10 There are ten gradients in total, and the sample volume is 5 μL.

[0078] The specific implementation method is as follows: (1) prepare 5 pairs of primer probe set solutions according to the concentration of probe 0.02mM and primer 0.03mM, prepare the diluted nucleic acid sample and the RT- The RAA fluorescence basic kit is ready for use, and...

Embodiment 3

[0112] Example 3 Sensitivity Verification of Detecting Novel Coronavirus Kit

[0113] The novel coronavirus detection kit provided by the present invention contains two parts: a reaction unit and a reaction buffer; the reaction buffer contains the novel coronavirus SARS-CoV-2 specific primer probe set determined in Example 2.

[0114] The reaction buffer is prepared by adding the specific primer probe set described in this application to the general reaction buffer in the F00R01 kit provided by Jiangsu Qitian Gene Biotechnology Co., Ltd. The components in the reaction buffer are: : upstream primer, downstream primer, probe, Tris-HCl buffer, MgAc and PEG 20000;

[0115] The reaction unit is the general reaction unit in the kit provided by Jiangsu Qitian Gene Biotechnology Co., Ltd. with the product number F00R01. It mainly includes the following components: ATP, dNTPs, recombinase, single-strand binding protein, exonuclease, DNA polymerase , reverse transcriptase.

[0116] Us...

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Abstract

The invention provides a primer probe set for detecting a novel corona virus SARS-CoV-2 and a detection method, and belongs to the technical field of molecular biology detection. The primer probe setfor detecting the novel corona virus SARS-CoV-2 by an RT-RAA fluorescence method comprises an upstream primer, a downstream primer and a probe, wherein the nucleotide sequence of the upstream primer is as shown in SEQID NO.1, the nucleotide sequence of the downstream primer is as shown in SEQID NO.2, and the probe is a substance, wherein the 31st-site basic group of a nucleotide sequence as shownin SEQID NO.3 is modified with a fluorescence report base group, the 32nd-site basic group of the nucleotide sequence as shown in SEQID NO.3 is replaced with a tetrahydrofuran residue, and the 34th-site basic group of the nucleotide sequence nucleotide sequence as shown in SEQID NO.3 is modified with a quenching base group. The primer probe set provided by the invention is quick, accurate and acute in detection, and simple and convenient to operate, the specificity can reach 100%, and detection of the novel corona virus SARS-CoV-2 can be completed within 30min.

Description

technical field [0001] The present invention relates to the technical field of virus detection, in particular to specific primers and probe sets for detecting novel coronavirus SARS-CoV-2, and the present invention also relates to methods and reagents for detecting novel coronavirus using the primers and probes box. Background technique [0002] On February 11, 2020, the Coronavirus Research Group (CSG) of the International Committee on Taxonomy of Viruses published the latest paper on the naming of new coronaviruses on the medical preprint publishing platform medRxiv, officially naming the new coronavirus from "2019-CoV" for "SARS-CoV-2". [0003] Coronaviruses have an envelope, and the particles are round or oval, often pleomorphic, with a diameter of 50-200nm. The S protein is located on the surface of the virus to form a rod-shaped structure. As one of the main antigenic proteins of the virus, it is the main gene used for typing. The N protein wraps the viral genome a...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/6844C12N15/11
CPCC12Q1/6844C12Q1/701C12Q2531/119C12Q2521/107C12Q2563/107Y02A50/30
Inventor 马学军郭利川申辛欣蔡禹希王佶顾赛艺何小周应清界王智宏
Owner JIANGSU QITIAN GENE BIOTECHNOLOGY CO LTD
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