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Nucleic acid composition for detecting novel coronavirus COVID-19 and application

A technology for COVID-19 and nucleic acid composition, applied in the field of nucleic acid composition for the detection of novel coronavirus COVID-19, can solve the problems of low sensitivity, complicated operation, long diagnosis time, etc. fast effect

Inactive Publication Date: 2020-07-03
NOVOPROTEIN SCI INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] Most of the existing technologies have disadvantages such as long diagnosis time, complicated operation and low sensitivity
The complex and changeable diagnostic environment limits the application of gene molecular detection technology, and also poses challenges to the development of technology

Method used

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  • Nucleic acid composition for detecting novel coronavirus COVID-19 and application
  • Nucleic acid composition for detecting novel coronavirus COVID-19 and application
  • Nucleic acid composition for detecting novel coronavirus COVID-19 and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0063] This embodiment provides a method for detecting the N gene of novel coronavirus COVID-19, comprising the following steps:

[0064] First, in order to detect the expression of the new coronavirus RNA, according to the nucleotide sequence of the N gene of the new coronavirus COVID-19 retrieved from the NCBI and Genbank databases, a conservative sequence of the N gene of the new coronavirus COVID-19 was chemically synthesized, and transfected Infect the cells, artificially prepare the pseudovirus, extract and serially dilute, and the diluted RNA is used as a template for transcription-mediated amplification (TMA).

[0065] Second, design and synthesize TMA upstream and downstream primers for amplifying the N gene of COVID-19 by selecting a highly conserved segment without secondary structure. Wherein, the sequence of the upstream primer N-t7F is: 5'-AATTTAATACGACTCACTATAGGGtgtaggtcaaccacgttccc-3'. Among them, capital letters are T7 promoter sequences, and lowercase letter...

Embodiment 2

[0073] The present embodiment provides a method for rapid detection and detection of the Orf1ab gene of novel coronavirus COVID-19, comprising the following steps:

[0074] First, pseudoviruses are artificially prepared as templates for transcription-mediated amplification (TMA).

[0075]Second, design and synthesize upstream and downstream primers for TMA amplification of the Orf1ab gene of COVID-19. Wherein, the upstream primer Orf1ab-t7F is: 5'-AATTTAATACGACTCACTATAGGGgcattctgtgaattataagg-3'. Among them, the 24 bases at the 5' end of Orf1ab-t7F are the added T7 phage promoter sequence, and the 20 bases at the 3' end are the specific sequence that hybridizes with the 5' end of the template RNA (+). The downstream primer Orf1ab-R is: 5'-atactgctgccgtgaacatg-3', which is a sequence hybridizing with RNA (-).

[0076] Third, by designing and synthesizing primers, using the method of annealing the primers in boiling water and in vitro transcription, a sgRNA targeting the negati...

Embodiment 3

[0083] This embodiment provides a method for detecting the Orf1ab gene of the new coronavirus COVID-19 with TMA and Cas13a "two-in-one", including the following steps:

[0084] First, the templates, primers and nucleic acid probes required in Example 3 can be prepared according to Example 2.

[0085] Second, establish a "two-in-one" reaction system (50 μL) of TMA combined with Cas13a. The reagents and specific concentrations involved in this reaction are: RNA (+) fragment to be detected, TMA upstream and downstream primers Orf1ab-t7F (1μM) and Orf1ab-R (1μM), 2000-4000U M-MLV reverse transcriptase, 500-2000U T7 RNA polymerase, 4U RNase inhibitor, 2μM cas13a protease, 400nM Orf1ab-sgRNA, 0.5μM OligoRNA-Cas13a-probe, TC reaction buffer (20-50mM Tris-HClpH7.5@25℃, 20-40mM KCl, 1- 5mM MgCl 2 , 1-5mM rNTPs, 1-5mM dNTPs, 20-50% glycerol, 0.5-1mMDTT).

[0086] Thirdly, after the components of the reaction system were gently vortexed and mixed, the reaction tube was reacted by a fl...

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Abstract

The invention discloses a nucleic acid composition for detecting novel coronavirus COVID-19 and application. The nucleic acid composition is prepared by the following steps: firstly, carrying out nucleic acid sample amplification based on a transcription-mediated amplification technology (TMA); then, specifically detecting amplified virus target nucleic acid in combination with activity of 'associated cleavage' of CRISPR-Cas13a protease; adding sgRNA, Cas13a protein, an ssRNA signal report probe and a reaction buffer solution into a reaction system containing target nucleic acid to be detected; and reading and detecting signals when the reaction is carried out so as to detect a target gene. By using the method disclosed by the invention, whether a sample contains a target nucleic acid sequence or not can be rapidly detected; and the nucleic acid composition is combined with the transcription-mediated nucleic acid amplification technology, so that the sensitivity of the detection methodcan reach a nanomole level, and the nucleic acid composition for detecting the novel coronavirus COVID-19 is suitable for rapidly detecting pathogenic microorganisms, gene mutation, specific target RNA and the like.

Description

technical field [0001] The invention belongs to the field of molecular biology technology, in particular to a nucleic acid composition and application for detecting novel coronavirus COVID-19. Background technique [0002] Since the outbreak of the new coronavirus COVID-19, the rapid spread of the epidemic has affected the hearts of the Chinese people and put unprecedented pressure on the national epidemic prevention and control. Most provinces and cities have entered the first-level response state of public health incidents. At present, there are 7 known human coronaviruses, namely HCoV-229E discovered in 1965, HCoV-OC43 identified in 1967, SARS-CoV that appeared in Guangdong, my country in 2003, and SARS-CoV that appeared in the Netherlands in 2004. HCoV-NL63 was isolated and identified, HCoV-HKU1 was screened in Hong Kong in 2005, MERS-CoV emerged in the Middle East in 2012, and the novel coronavirus COVID-19 was discovered in Wuhan, China in 2019. Among them, the new cor...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/6844C12N15/11
CPCC12Q1/6844C12Q1/701C12Q2521/327Y02A50/30
Inventor 王笃强位小丫赵曼曼李瑞霞郑实
Owner NOVOPROTEIN SCI INC
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