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Preparation method of pseudotype virus for nucleic acid detection of 2019-nCoV

A virus nucleic acid, pseudovirus technology, applied in the field of pseudovirus preparation, can solve complex problems

Active Publication Date: 2020-07-31
复百澳(苏州)生物医药科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] In the outbreak of the 2019 novel coronavirus, the final diagnosis of the disease requires nucleic acid detection in the laboratory, and the actual nucleic acid detection experiment process is relatively complicated, including the collection of patient samples, virus nucleic acid extraction, RNA reverse CDNA and QPCR detection and other experimental links

Method used

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  • Preparation method of pseudotype virus for nucleic acid detection of 2019-nCoV
  • Preparation method of pseudotype virus for nucleic acid detection of 2019-nCoV
  • Preparation method of pseudotype virus for nucleic acid detection of 2019-nCoV

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0080] Example 1: Construction of a pseudoviral vector that eliminates unnecessary elements of the lentiviral expression vector

[0081] In the existing lentiviral vector FV011 (Fubai’s own vector, such as Picture 8 Shown), remove the promoter and foreign expression box:

[0082] Step S011: Enzyme digestion of the lentiviral vector at 37°C in a water bath for 2 hours. The digestion system is specifically:

[0083]

[0084] Step S012: Agarose gel electrophoresis and gel recovery target product: use 1% agarose, 120V constant pressure electrophoresis for 30-40 minutes, cut out a single target fragment under ultraviolet irradiation and put it into a clean centrifuge tube for gel recovery and purification. Loading information: samples 8-9: FV-011-PacI-NheI; the electrophoresis results are as follows figure 1 Shown.

Embodiment 2

[0085] Example 2: Cloning part of the genome of the virus to be detected into a pseudovirus vector;

[0086] Step S021: Obtain the partial sequence of the coronavirus by way of full gene synthesis, and synthesize it into the PUC57 vector, denoted as PUC57-ORF 1a / bEN; the synthetic sequence contains the PAC I restriction site at the 5'end and the Nhe I enzyme at the 3'end The cut site, the specific sequence information is as follows:

[0087] TAATTAAATCGTGTTGTCTGTACTGCCGTTGCCACATAGATCATCCAAATCCTAAAGGATTTTGTGACTTAAAAGGTAAGTATGTACAAATACCTACAACTTGTGCTAATGACCCTGTGGGTTTTACACTTAAAAACACAGTCTGTACCGTCTGCGGTATGTGGAAAGGTTATGGCTGTAGTTGTGATCAACTCCGCGAACCCATGCTTCAGTCAGCTGATGCACAATCGTTTTTAAACGGGTTTGCGGTGTAAGTGCAGCCCGTCTTACACCGTGCGGCACAGGCACTAGTACTGATGTCGTATACAGGGCTTTTGACATCTACAATGATAAAGTAGCTGGTTTTGCTAAATTCCTAAAAACTAATTGTTGTCGCTTCCAAGAAAAGGACGAAGATGACAATTTAATTGATTCTTACTTTGTAGTTAAGAGACACACTTTCTCTAACTACCAACATGAAGAAACAATTTATAATTTACTTAAGGATTGTCCAGCTGTTGCTAAACATATGTACTCATTCGTTTCGGAAGAGACAGGTACGTTAATAGTTA...

Embodiment 3

[0106] Example 3: The integrase gene of the lentivirus packaging helper plasmid is mutated to reduce or lose the integration ability of the lentivirus:

[0107] Before mutation:

[0108] tttttagatggaatagataaggcccaagaagaacatgagaaatatcacagtaattggagagcaatggctagtgattttaacctaccacctgtagtagcaaaagaaatagtagccagctgtgataaatgtcagctaaaaggggaagccatgcatggacaagtagactgtagcccaggaatatggcagcta Gat tgtacacatttagaaggaaaagttatcttggtagcagttcatgtagccagtggatatatagaagcagaagtaattccagcagagacagggcaagaaacagcatacttcctcttaaaattagcaggaagatggccagtaaaaacagtacatacagacaatggcagcaatttcaccagtactacagttaaggccgcctgttggtgggcggggatcaagcaggaatttggcattccctacaatccccaaagtcaaggagtaatagaatctatgaataaagaattaaagaaaattataggacaggtaagagatcaggctgaacatcttaagacagcagtacaaatggcagtattcatccacaattttaaaagaaaaggggggattggggggtacagtgcaggggaaagaatagtagacataatagcaacagacatacaaactaaagaattacaaaaacaaattacaaaaattcaaaattttcgggtttattacagggacagcagagatccagtttggaaaggaccagcaaagctcctctggaaaggtgaaggggcagtagtaatacaagataatagtgacataaaagtagtgcca AGaAGaAaA gcaaagatcatca...

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Abstract

The invention discloses a preparation method of a pseudotype virus for nucleic acid detection of 2019-nCoV, and relates to the technical field of biology. The preparation method comprises a method forpreparing standard quality control products which contain but are not limited to a 2019-nCoV nucleotide sequence and are used for nucleic acid detection through a non-integrated slow virus vector system, a method which is used for preparing the standard quality control products for nucleic acid detection, and a method for constructing a non-integrated slow virus through mutating 64-locus amino acid Asp of integrase of slow virus auxiliary plasmid into Asn, and amino acids Arg, Arg and Lys at 262-264 loci into Ala, Ala and His. In the manner of removing elements of promoters and the like of slow viruses, the load quantity of virus vectors can be promoted, a pseudotype virus which can be used for nucleic acid detection and complete flow quality control is constructed, and the preparation method can be used for constructing a pseudotype virus for corona viruses and can also be used for constructing a pseudotype virus for SARS-COV, MERS-COV, influenza viruses and the like.

Description

Technical field [0001] The present invention belongs to the field of biotechnology, and in particular relates to a method for preparing a pseudovirus for nucleic acid detection of 2019 new coronavirus. Background technique [0002] The 2019 Novel Coronavirus (2019-NCOV) is known to cause colds and more serious diseases such as Middle East Respiratory Syndrome (MERS) and Severe Acute Respiratory Syndrome (SARS). The diameter of the coronavirus is about 80-120NM, the 5'end of the genome has a methylated cap structure, and the 3'end has a POLY(A) tail. The total length of the genome is about 27-32KB. It is currently the largest virus in the genome of RNA viruses. . [0003] In the 2019 new crown virus outbreak, the final diagnosis of the disease requires laboratory nucleic acid testing. The actual nucleic acid testing experiment process is more complicated, including the collection of patient samples, viral nucleic acid extraction, RNA reverse cDNA and QPCR testing and other experime...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/867C12N15/50C12N7/01
CPCC07K14/005C12N7/00C12N15/86C12N2740/15043C12N2770/20021C12N2770/20022
Inventor 刘骏周春艳金丽谢伟建徐金凤
Owner 复百澳(苏州)生物医药科技有限公司
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