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In-vitro amplification detection method for novel coronavirus nucleic acid and test kit thereof

A viral nucleic acid, in vitro amplification technology, applied in microorganism-based methods, biochemical equipment and methods, and microbial determination/inspection, etc., can solve the problems of PCR product contamination, complicated operation, false positives, etc., and reduce PCR products. Contamination, high sensitivity, and the effect of improving specificity

Inactive Publication Date: 2020-07-10
成都因泰生命科技有限公司
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Problems solved by technology

First use the outer primer to amplify, and then take the amplified product as a template and use the inner primer to amplify, that is, to perform two consecutive PCR reactions. This method is more complicated and time-consuming, and each PCR In , the efficiency of amplification will not exceed 2 n At the same time, because the second amplification needs to use the PCR product amplified by the outer primer as the template, it is easy to cause contamination of the PCR product, resulting in false positive amplification.

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  • In-vitro amplification detection method for novel coronavirus nucleic acid and test kit thereof

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Embodiment 1

[0022] 1. Materials

[0023] Human peripheral blood DNA was extracted by the conventional phenol-chloroform extraction method in our laboratory, and used as the template to be amplified.

[0024] 2. Primer design: Synthesize a pair of specific outer primers and a pair of special nested primers and complementary primers of the nested primers according to the sequence of the new coronavirus nucleic acid ORF1ab gene, and synthesize the following specific inner and outer primers:

[0025] Outer upstream primer P1: 5'-ACAACTTGTGCTAAT-3' (SEQ ID NO: 1);

[0026] Outer downstream primer P2: 5'-CATCAGCTGACTGAAG-3' (SEQ ID NO: 2);

[0027] Inner upstream primer P3: 5'-TTTTCCCTGTGGGTTTTACACTT-3' (SEQ ID NO: 3);

[0028] Inner downstream primer P4: 5'-TTTTTGGGTTCGCGGAGTTGAT-3' (SEQ ID NO: 4).

[0029] Complementary primer P3C of the inner upstream primer P3: 5'-AGTGTAAAACCCACAGG-3' (SEQ ID NO: 5);

[0030] Complementary primer of the inner downstream primer P4C: 5'-TCAACTCCGCGAACC-3'...

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Abstract

The invention discloses an in-vitro amplification detection method for novel coronavirus nucleic acid and a test kit thereof. The method comprises the following steps: (1) extracting to-be-detected DNA; and (2) taking the to-be-detected DNA as a template, and carrying out reverse transcription NCA-QPCR by adopting primers as shown in SEQ ID NO. 1-6. The method provided by the invention can effectively solve the efficiency of specific amplification and the inhibition effect of non-specific amplification on specific amplification, and can rapidly, accurately and sensitively detect the novel coronavirus nucleic acid.

Description

technical field [0001] The invention belongs to the technical field of gene detection, and in particular relates to a method for in vitro amplification detection of a new coronavirus nucleic acid and a kit thereof. Background technique [0002] The existing in vitro nucleic acid amplification detection technology and its kits for the new coronavirus are basically based on the RT-QPCR technology platform. This technology firstly synthesizes template cDNA by reverse transcription of RNA, and then synthesizes heat-resistant DNA polymerase, specific primer probe , dNTPs substrate, template cDNA, magnesium ions, etc. are placed in the same buffer reaction system, and thermal cycles of high temperature, low temperature, and medium temperature are repeated to denature the template DNA, anneal the primer and the template, and extend the primer repeatedly. Fluorescent detection, while reaching the target DNA fragments in vitro showed 2 n Double amplification (where n is the number o...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/6848C12N15/11C12R1/93
CPCC12Q1/6848C12Q1/701C12Q2521/107C12Q2531/113C12Q2549/119
Inventor 刘文涛赖演媚
Owner 成都因泰生命科技有限公司
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