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Reagent and method for detecting novel coronavirus

A new type of coronavirus technology, applied in the field of clinical medical nucleic acid molecular detection, can solve problems such as high biosafety risks, unstable quality, and destruction

Pending Publication Date: 2021-10-01
苏州中科细胞转化研究院
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

But both have obvious disadvantages: 1. The quality of RNA transcribed in vitro is unstable when used as a quality control product, and it is easily damaged by RNase in the environment; the sample processing process cannot be effectively monitored; 2. Clinically positive viruses are used as quality control products , there is a high biosafety risk for highly pathogenic viruses (eg: new coronavirus, SARS, MERS, etc.)

Method used

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  • Reagent and method for detecting novel coronavirus
  • Reagent and method for detecting novel coronavirus
  • Reagent and method for detecting novel coronavirus

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preparation example Construction

[0095] This paper also includes the preparation method of the above-mentioned pseudovirus, including introducing the nucleic acid construct containing the nucleic acid molecule described herein into the engineered cell, and cultivating the engineered cell under the conditions of transcription and / or translation of the nucleic acid construct, and optionally by said Engineered cells obtain and purify pseudoviral particles. Those skilled in the art are aware of common engineering cell culture methods, and methods for obtaining and purifying virus particles from engineered cells, such as sonication, organic solvent precipitation, and the like.

[0096]For example, in the embodiment of the novel coronavirus positive pseudovirion (positive control), the preparation process of the present invention includes: (1) obtaining ORF1ab gene, N gene and E gene sequence for novel coronavirus, such as sequence SEQ ID NO Shown in: 1; Described ORF1ab gene, N gene and E gene are obtained by synt...

Embodiment 1

[0149] Embodiment 1, the construction of pseudovirus quality control product expression vector

[0150] 1. The construction of the pseudovirus expression vector pET32a-MS2-SARS-COV2-ORF1ab+N+E of three genes (ORF1ab+N+E):

[0151] Refer to the MS2 phage genome sequence (Reference Sequence: NC_001417.2) in the NCBI database and the ORF1ab gene, N gene, and E gene of SARS-Cov-2 (Novel coronavirus nucleic acid detection published by the Chinese Center for Disease Control and Prevention of Viral Disease Prevention and Control) Primer and probe sequence coverage area), prepared by artificial synthesis, including the MS2 phage maturation enzyme coding gene, capsid protein coding gene, packaging site sequence, cDNA sequence corresponding to the new coronavirus nucleic acid fragment and auxiliary enzyme cutting site The auxiliary multiple cloning site is located in the middle of the ORF1ab, N and E gene fragments. The MS2 phage sequence is shown in SEQ ID NO: 2 in the sequence table,...

Embodiment 2

[0156] Embodiment 2, the preparation of pseudovirus quality control product

[0157] (1) Transform the plasmid expression vector prepared in Example 1 into Escherichia coli BL21 competent cells, spread it on a nutrient agar plate containing ampicillin (final concentration is 50 μg / ml), and culture it at 37°C for 18h (nutrient agar The specific preparation method of the plate is as follows: Tryptone 1g, NaCl 0.5g, yeast extract 0.5g, agar powder 1.5g, dissolved in 90mL ddH 2 O, adjust the pH to 7.0-7.2, then add ddH 2 O and dilute to 100mL, autoclaved for later use. Then pick positive monoclonal bacteria and inoculate them in LB liquid medium containing ampicillin for overnight culture.

[0158] (2) Inoculate 2ml of overnight cultured bacterial liquid into 200ml of ampicillin-resistant LB liquid medium (final concentration of ampicillin is 100μg / ml), shake at 200rpm until the OD value is about 0.6-0.8 (liquid LB medium The specific preparation method is: tryptone 1g, NaCl 0....

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Abstract

The invention discloses a pseudovirus quality control product of a novel coronavirus (SARS-Cov-2) nucleic acid detection method and the application of the pseudovirus quality control product, and belongs to the technical field of clinical medicine nucleic acid molecule detection. according to the invention, SARS-Cov-2 characteristic gene (ORF1ab, N and E) fragments are prepared into a plurality of pseudoviruses, and a positive pseudovirus quality control product system is formed. Respective characteristic gene fragments of other highly pathogenic human coronaviruses (SARS, MERS, HKU1, OC43, NL63 and 229E) are prepared into a series of pseudoviruses, and a negative pseudovirus quality control product system is formed. When nucleic acid molecule detection is carried out on SARS-Cov-2 in clinical medicine, the positive pseudovirus quality control product, the negative pseudovirus quality control product and a patient sample to be detected participate in the whole experiment operation process including sample treatment together, so that false negative or false positive experiment results are effectively prevented from being generated. according to the application of the pseudovirus quality control product system prepared by the invention, the biological safety risk caused by high pathogenic microorganisms in the research, development and verification process of an SARS-Cov-2 nucleic acid molecule detection method is reduced to the greatest extent.

Description

technical field [0001] The invention belongs to the technical field of nucleic acid molecular detection in clinical medicine, and in particular relates to reagents and methods for detecting novel coronaviruses. Background technique [0002] The 2019 novel coronavirus (SARS-Cov-2) is an enveloped, non-segmented positive-sense single-stranded RNA virus with round or oval particles and a diameter of about 60-140nm, belonging to the order Reticuloviridae Coronaviridae. Each genome of this virus is about 30,000 nucleotides in length, encoding four structural proteins: S protein (spike protein), M protein (membraneglycoprotein), nucleocapsid protein N (nucleocapsid phosphoprotein), and small envelope E ( envelope protein) protein. Its gene sequence shows that SARS-Cov-2 belongs to a virus with a long branch in the evolutionary tree of Betacoronavirus Lineage β (Betacoronavirus Lineage β, Sarbecovirus), such as MERS-CoV or SARS-CoV. [0003] In the "Diagnosis and Treatment Progr...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N7/00C12N15/50C12N15/70C12N1/21C12Q1/70C12R1/93
CPCC12N7/00C07K14/005C12N15/70C12Q1/701C12N2770/20021C12N2770/20022C12N2770/20052
Inventor 喻长杰于安路陈飞郭子平
Owner 苏州中科细胞转化研究院
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