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CRISPR-Cas13 a-based bovine viral diarrhea virus detection method

A technology of bovine viral diarrhea and virus, which is applied in the field of detection of bovine viral diarrhea virus, can solve the problems of high cost, difficult to deal with on-site and large-scale detection, and high probability of false positive, achieving high application value and high accuracy , the effect of rapid detection

Active Publication Date: 2020-11-24
SHIHEZI UNIVERSITY
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  • Abstract
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  • 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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  • CRISPR-Cas13 a-based bovine viral diarrhea virus detection method
  • CRISPR-Cas13 a-based bovine viral diarrhea virus detection method
  • CRISPR-Cas13 a-based bovine viral diarrhea virus detection method

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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 clarifying the present invention, not for limiting the scope of the present invention.

[0028] The inventor expresses and purifies LwCas13a protein with LwCas13a as the research center, and at the same time, designs 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 RNA-cleaving activity in vitro, and can specifically and accurately detect BVDV, and the invention was completed on this basis.

[0029] Step 1: Primer design for the detection method of bovine viral diarrhea virus based on CRISPR-Cas13a

[0030] 1. LwCas13a gene primer design

[0031] The full-length gene sequence of pC013-Twinstrep-SUMO-LwCas13a was obtained on addgene, and the full-length primers of LwCas13a protein were designed with Premier5 software. Th...

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Abstract

The invention discloses a primer for detecting bovine viral diarrhea virus, a kit prepared by using the primer and used for detecting BVDV, and a CRISPR-Cas13 a-based bovine viral diarrhea virus detection method. The primer is 5'-GGGGAUUUAGACUACCCCAAAAACGAAGGGGACUAAAACGCCAUCCAACGAACUCACCACUGUUGCU-3'; the kit at least comprises the primer; a sample to be detected is added into the kit for detection. According to the CRISPR-Cas13 a-based bovine viral diarrhea virus detection method, a CRISPR-Cas13a system is successfully applied to nucleic acid detection of the BVDV for the first time, and whether the BVDV is contained or not can be identified only by adding a micro-scale sample. The CRISPR-Cas13 a-based bovine viral diarrhea virus detection method has high specificity and sensitivity. The method provides a wide prospect for early diagnosis of the 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 contagious disease caused by Bovine Viral Diarrhea Virus (BVDV). It mainly infects cattle, but also other organisms such as pigs, sheep, and camels. wide. BVDV is a positive-sense ribonucleic acid (RNA) virus belonging to the family Flaviviridae and the genus Pestivirus. 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 diarrhea virus (BVDV) prevalences in the global cattle population [J]. Scientific reports, 2018, 8(1):1-15.). Among them, acute or chronic mucosal diseases, diarrhea, growth and reproduction disorders, immunosuppression and persistent infection...

Claims

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Application Information

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Patent Type & Authority Applications(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
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