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Novel coronavirus nucleic acid pseudovirus standard substance for detection and preparation method thereof

A coronavirus and standard substance technology, applied in the field of genetic engineering, can solve problems such as poor stability, detection error, and inability to replace the virus detection process, and achieve good stability and safety.

Active Publication Date: 2021-02-12
NAT INST OF METROLOGY CHINA
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] 1) Poor stability
[0007] The existing nucleic acid standard material of the new coronavirus is a standard material in the state of naked RNA, which is easily degraded by RNase in the environment, resulting in detection errors;
[0008] 2) Unable to simulate the nucleic acid extraction process of the new coronavirus
The naked RNA standard cannot replace the real virus detection process. For example, the nucleic acid extraction efficiency is only 70%, and it is impossible to evaluate the extraction efficiency by directly using naked RNA detection.
[0010] 3) Strict storage requirements for reference materials
[0011] Because it is naked RNA, it must be stored below -70°C, which is inconvenient for grassroots units

Method used

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  • Novel coronavirus nucleic acid pseudovirus standard substance for detection and preparation method thereof
  • Novel coronavirus nucleic acid pseudovirus standard substance for detection and preparation method thereof
  • Novel coronavirus nucleic acid pseudovirus standard substance for detection and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0085] Embodiment 1 novel coronavirus pseudovirus preparation scheme one

[0086] In this example, the lentiviral particle packaging gene sequence 1 (ORF1ab-1 gene), gene sequence 2 (E gene) and gene sequence 3 (N gene) fragment RNA was prepared by the following method:

[0087] 1.1 Cloning gene sequence 1 (ORF1ab-1 gene), gene sequence 2 (E gene) and gene sequence 3 (N gene) fragments by PCR from 2019-nCoV (or SARS-CoV-2) genome cDNA ( figure 1 ) and linearize the pCDH-CMV-MCS-EF1-puro vector plasmid, the primers are as follows (Table 1):

[0088] Table 1 Primer sequences of gene sequence 1, gene sequence 2 and gene sequence 3

[0089]

[0090] 1.2 Through the design of primers, each fragment of gene sequence 1, gene sequence 2 and gene sequence 3 of 2019-nCoV (or SARS-CoV-2) and the linearized vector plasmid have 20bp repeat sequences at both ends in order for subsequent Homologous recombination join;

[0091] 1.3 Perform agarose gel recovery and purification of the cl...

Embodiment 2

[0106] Embodiment 2 novel coronavirus pseudovirus preparation scheme two

[0107] In this embodiment, the lentiviral particle packaging gene sequence 1 (ORF1ab-1 gene) and gene sequence 3 (N gene) fragment RNA was prepared by the following method:

[0108] 2.1 Clone gene sequence 1 (ORF1ab-1 gene) and gene sequence 2 (N gene) fragments by PCR from 2019-nCoV (or SARS-CoV-2) genome cDNA and linearize the pLVX vector plasmid. See Table 1 for primers :

[0109] 2.2 Through the design of primers, each fragment of gene sequence 1 and gene sequence 3 of 2019-nCoV (or SARS-CoV-2) and the linearized vector plasmid have a 20bp repeat sequence at both ends in order for subsequent homologous recombination connection ;

[0110] 2.3 Perform agarose gel recovery and purification of the cloned gene fragments and linearized vector plasmids;

[0111] 2.4 Connect the gene sequence 1 and gene sequence 3 fragments of 2019-nCoV (or SARS-CoV-2) into one fragment by fusion PCR method;

[0112] 2....

Embodiment 3

[0123] Embodiment 3 novel coronavirus pseudovirus preparation scheme three

[0124] In this embodiment, the fragment RNA of lentiviral particle packaging gene sequence 3 (N gene) is prepared by the following method:

[0125] 3.1 From the 2019-nCoV (or SARS-CoV-2) genome cDNA, clone the gene sequence 3 (N gene) fragment by PCR and linearize the pBoBi vector plasmid. The primer sequences are shown in Table 1:

[0126] 3.2 Through the design of primers, each fragment of gene sequence 1, gene sequence 2 and gene sequence 3 of 2019-nCoV (or SARS-CoV-2) and the linearized vector plasmid have 20bp repeat sequences at both ends in order for subsequent Homologous recombination join;

[0127] 3.3 Perform agarose gel recovery and purification of the cloned gene fragments and linearized vector plasmids;

[0128] 3.4 Connect each fragment of the gene sequence 3 of 2019-nCoV (or SARS-CoV-2) into one fragment by fusion PCR method;

[0129] 3.5 Take the PCR tube, add the 2019-nCoV (or SARS...

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Abstract

The invention relates to a pseudovirus standard substance for nucleic acid detection of a novel coronavirus (2019-nCoV or called SARS-CoV-2) and a preparation method of the pseudovirus standard substance. Novel coronavirus nucleic acid is integrated into a lentiviral expression plasmid vector, then constructed lentiviral expression plasmids and packaging plasmids are jointly transfected into cells, and a large number of pseudoviral particles packaged with novel coronavirus RNA are obtained after cell expression. After plasmid DNA is removed through DNase I digestion and sucrose density gradient ultracentrifugation, freeze drying is performed to obtain a pseudovirus particle freeze-dried product. The pseudovirus standard substance has the partial RNA sequence packaged with the virus and also has a pseudovirus standard substance packaged with all the sequences, can completely simulate the whole process of virus RNA extraction and nucleic acid detection, and can be used for the verification evaluation and the laboratory quality control of the novel coronavirus nucleic acid qualitative and quantitative detection method. The obtained standard substance has the characteristics of high purity, good safety, accurate quantity value and the like, is good in stability, can be transported at normal temperature, and provides guarantee for quantity value traceability and transmission of thestandard substance.

Description

Technical field: [0001] The invention relates to the technical field of genetic engineering, in particular to a standard substance for detecting novel coronavirus nucleic acid pseudoviruses and a preparation method thereof. Background technique: [0002] At present, the novel coronavirus (2019-nCoV, also known as SARS-CoV-2) infection is a global pandemic, and there is an urgent need for continuous detection of a large number of people infected with the novel coronavirus. [0003] Polymerase chain reaction (PCR) is considered to be the "gold standard" for the diagnosis of patients with new coronavirus pneumonia. However, the quality of PCR detection reagents for new coronavirus is uneven, which has a great impact on the detection of new coronavirus and needs to be resolved urgently. [0004] In order to be able to standardize the quality control of the detection PCR reagents produced by various manufacturers, the National Institute of Metrology of China urgently developed tw...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N7/00C07K14/155C12Q1/70C12Q1/686
CPCC12N7/00C07K14/005C12Q1/701C12Q1/686C12N2770/20021C12N2740/15022C12Q2563/159Y02A50/30
Inventor 隋志伟戴新华彭珂张永卓方向黄文锋
Owner NAT INST OF METROLOGY CHINA
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