Dual-target site inverse-transcription fluorescent PCR primers, probes and kits for detection of 2019 new coronavirus

A coronavirus, primer-probe technology, applied in biochemical equipment and methods, microbial determination/inspection, resistance to vector-borne diseases, etc., can solve problems such as false negative test results, missed detection, etc.

Pending Publication Date: 2020-04-28
西安博睿康宁生物医学中心有限公司
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0004] 2019-nCoV is a new type of SARS mutant virus, but there is currently no commercial 2019-nCoV nucleic acid detection kit certified by CFDA. Some biotechnology companies have developed nucleic acid detection kits for scientific research for 2019-nCoV detection
Therefore, for nucleic acid detection of viruses, a single target site is prone to false negative detection results due to mutations, resulting in missed detection

Method used

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  • Dual-target site inverse-transcription fluorescent PCR primers, probes and kits for detection of 2019 new coronavirus
  • Dual-target site inverse-transcription fluorescent PCR primers, probes and kits for detection of 2019 new coronavirus
  • Dual-target site inverse-transcription fluorescent PCR primers, probes and kits for detection of 2019 new coronavirus

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 12019

[0042] Example 1 2019 Novel Coronavirus 2019-nCoV Gene Sequence Alignment and Target Sequence Determination

[0043] According to the newly published 6 2019-nCoV whole genomes, the nucleic acid sequence was compared through the NCBI database, and the specific nucleic acid sequence in the N gene of the virus was found: W1 (SEQ ID NO.1), W2 (SEQ ID NO.2) and W3 (SEQ ID NO. 3).

Embodiment 2

[0044] Embodiment 2 is aimed at the design of the primer of target sequence and probe

[0045] For the target sequence (SEQ ID NO.1-3) determined in Example 1, the inventors designed multiple combinations of primers and probes. And further, from many primer-probe combinations, 4 groups of MGB probe and primer combinations with better detection effects were selected, as shown in Table 1-Table 4 respectively. System 1 is used to detect target sequence W1, system 2 is used to detect target sequence W2, and systems 3 and 4 are used to detect target sequence W3, respectively.

[0046] Table 1 Primer Probe Sequence of System 1

[0047] 2019-nCoV-F1: TGGCAATGGCGGTGATG (SEQ ID NO.4) 2019-nCoV-R1: AGCTGGTTCAATCTGTCAAGCA (SEQ ID NO.5) 2019-nCoV-P1: TGCTCTTGCTTTGCTG (SEQ ID NO.6)

[0048] Primer probe sequence of table 2 system 2

[0049] 2019-nCoV-F2:GAAGCCTCGGCAAAAACG (SEQ ID NO.7) 2019-nCoV-R2:GCCGAAAGCTTGTGTTACATTG (SEQ ID NO.8) 2019-nCo...

Embodiment 3

[0064] Example 3 Verification of the detection sensitivity and specificity of 4 fluorescent PCR detection systems for 2019-nCoV

[0065] 1. Sensitivity evaluation

[0066] At present, due to the lack of 2019-nCoV strains, the sensitivity experiment uses the RNA sequence of the artificially synthesized 2019-nCoV sequence after in vitro reverse transcription as a positive template, in which the SARS synthetic fragments: S1-S3, respectively correspond to W1-W3 of 2019-nCoV, The sequences of S1-S3 are shown in SEQ ID NO.16-18 respectively; MERS synthetic fragments: M1-M3, respectively corresponding to W1-W3 of 2019-nCoV, and the sequences of M1-M3 are shown in SEQ ID NO.19-21 respectively Show. The synthetic sequences were mixed into 12 normal human throat swabs, and the detection results of 4 sets of RT-qPCR were all positive, with a sensitivity of 100% (12 / 12).

[0067] 2. Specificity evaluation

[0068] Use the 4 sets of RT-qPCR screened and determined in Example 2 to detect...

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Abstract

The invention provides dual-target site inverse-transcription fluorescent PCR primers, probes and kits for detection of 2019 new coronavirus (2019-nCoV). Nucleotide sequence alignment is performed onthe whole genome of the 2019-nCoV, 3 specific nucleotide sequences W1, W2 and W3 are found in N genes of the virus, and are shown in SEQ ID No. 1-3, multiple pairs of primer probes are designed separately for three target sites, and it is found that when a primer probe combination which is designed for the target sites of W1 and W2 and has sequences shown in SEQ ID No. 4-6 and SEQ ID No. 7-9 is applied to double-target site inverse-transcription fluorescent PCR, 2019-nCoV can be detected specifically and sensitively with detection sensitivity of within 10 copies; and no non-specific amplification of 22 clinically-common respiratory pathogens are caused, false-negative results caused by single target spot mutation can be avoided effectively, and good detection ability of specimens is achieved.

Description

technical field [0001] The present invention relates to the technical field of virus detection, in particular to target sites for detecting 2019 novel coronavirus, specific primers and probes for the target sites, and the present invention also relates to the use of the primers and probes to detect 2019 novel coronavirus Detection methods and kits. Background technique [0002] Novel coronavirus (2019-nCoV) is the latest human-pathogenic coronavirus detected in January 2020, and it is the respiratory pathogen that causes pneumonia outbreaks of unknown cause. The clinical manifestations are fever, fatigue and other systemic symptoms, accompanied by dry cough, and dyspnea is common in hospitalized patients; the vital signs of most patients are generally stable when they are admitted to the hospital. Although the virus has not been found to be contagious between humans, it still requires close monitoring to prevent larger infections. From January 11th to 12th, 2020, six sets ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/6851C12N15/11
CPCC12Q1/6851C12Q1/701C12Q2600/166C12Q2531/113C12Q2521/107C12Q2563/107C12Q2545/113Y02A50/30
Inventor 李睿周梦诗
Owner 西安博睿康宁生物医学中心有限公司
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