Novel coronavirus nucleic acid detection kit, preparation method and application

Abstract

The invention discloses a novel coronavirus nucleic acid detection kit as well as a preparation method and application thereof. The novel coronavirus nucleic acid detection kit comprises primers and probes synthesized according to novel coronavirus nucleic acid ORF1ab gene, N gene and E gene sequences, a pair of specific internal control primers and TaqMan probe ACE2TM synthesized according to anACE2 gene sequence, reverse transcriptase, heat-resistant DNA polymerase, dNTPs, magnesium ions, a PCR buffer solution, ultrapure water and an RNA template to be detected; the method comprises the following steps: performing reverse transcription reaction on a buffer reaction system by a one-step method to synthesize template cDNA, performing repeated thermal cycle to obtain a PCR product, degrading a probe release fluorescein A of the PCR product, and degrading a probe release fluorescein B of the ACE2 gene. The PCR product guided by the inner primer is increased by more than two times, so that the efficiency of specific amplification can be fundamentally improved, and a false negative result caused by a sampling error can be identified, thereby improving the accuracy of a detection result.

Description

technical field [0001] The invention relates to the field of RNA in vitro amplification technology in molecular biology detection technology, in particular to a novel coronavirus nucleic acid detection kit, preparation method and application. 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 first reverse-transcribes RNA to synthesize template cDNA, and then synthesizes heat-resistant DNA polymerase, specific primer probe, dNTPs substrates, 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 for thermal cycling and fluorescence. Detection, and the target DNA fragment is amplified by 2n times in vitro...

AI Technical Summary

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 to operate and takes a long time, and each PCR In addition, the efficiency of amplification will not exceed 2n times. At the same time, because the second amplification needs to use the PCR product amplified by the outer primer as a template, it is easy to cause PCR product contamination, resulting in false positive amplification.

[0006] In addition, because the new coronavirus mainly interferes with the deep alveolar epithelial cells, when the virus titer is low, the virus or cells containing the virus cannot be obtained due to non-standard sampling techniques and methods, and false negative results may also be produced, thereby reducing the detection capacity. accuracy of results

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