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Detection method of bovine viral diarrhea virus based on CRISPR-Cas13a

A bovine viral diarrhea and virus technology, which is applied in the field of bovine viral diarrhea virus detection, can solve the problems of difficult on-site and large-scale testing, high cost, and high probability of false positives, and achieves high accuracy and high application value. , the effect of rapid detection

Active Publication Date: 2022-06-07
SHIHEZI UNIVERSITY
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, the traditional BVDV methods almost all require sophisticated instruments and high costs, which are difficult to deal with on-site and large-scale detection, and the probability of false positives is relatively high (Adams M J, Hendrickson R C, Dempsey D M, et al.Tracking the changes in virustaxonomy[J].Archives of virology,2015,160(5):1375-1383.)

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  • Detection method of bovine viral diarrhea virus based on CRISPR-Cas13a
  • Detection method of bovine viral diarrhea virus based on CRISPR-Cas13a
  • Detection method of bovine viral diarrhea virus based on CRISPR-Cas13a

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Embodiment Construction

[0027] The present invention will be further described in detail below in conjunction with specific embodiments, and the given examples are only for illustrating the present invention, rather than for limiting the scope of the present invention.

[0028] The inventors took LwCas13a as the research center to express and purify the LwCas13a protein, and at the same time, designed a specific crRNA according to the 5'UTR conserved region of the reported BVDV gene sequence. The RNA obtained from BVDV was used to verify that Cas13a also has RNase cleavage activity in vitro, and BVDV can be detected specifically and accurately. On this basis, the invention was completed.

[0029] The first step: primer design for the detection method of bovine viral diarrhea virus based on CRISPR-Cas13a

[0030] 1. Primer design of LwCas13a gene

[0031] The full-length gene sequence of pC013-Twinstrep-SUMO-LwCas13a was obtained on addgene, and the LwCas13a protein full-length primer was designed wi...

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Abstract

The invention discloses a primer for detecting bovine viral diarrhea virus, a kit for detecting BVDV prepared by using the primer, and a detection method for bovine viral diarrhea virus based on CRISPR-Cas13a. '; The kit contains at least the above primers, and the sample to be tested is added to the kit for detection. In the present invention, for the first time, the CRISPR-Cas13a system is successfully used in the nucleic acid detection of BVDV virus, and only a small amount of sample is added to identify whether it contains BVDV virus. Has good specificity and sensitivity. This method provides broad prospects for the early diagnosis of BVDV.

Description

technical field [0001] The invention relates to the technical field of biological detection, in particular to a detection method for bovine viral diarrhea virus based on CRISPR-Cas13a. Background technique [0002] Bovine viral diarrhea (mucosal disease) is a contact infectious disease caused by Bovine Viral Diarrhea Virus (BVDV), which mainly infects cattle, but also infects pigs, sheep, camels and other organisms. wide. BVDV is a positive-sense ribonucleic acid (RNA) virus belonging to the Flaviviridae and Pestivirus genus. Infection can lead to a variety of clinical manifestations, including respiratory, gastrointestinal, and reproductive (various forms) disorders ( B, Roch F F, Richter V, et al. A meta-analysis of bovine viral diarrhoea virus (BVDV) prevalences in the global cattle population [J]. Scientific reports, 2018, 8(1): 1-15.). Among them, the symptoms of acute or chronic mucosal disease, diarrhea, growth and reproduction disorders, immunosuppression and pe...

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/70C12Q1/682C12N15/11
CPCC12Q1/701C12Q1/682C12Q2563/107C12Q2521/327
Inventor 倪伟胡圣伟姚瑞李村院李晓悦徐越仁
Owner SHIHEZI UNIVERSITY