Triple RPA (recombinase polymerase amplification) detection kit for bovine viral diarrhea virus, bovine coronavirus and bovine rotavirus

A technology for bovine viral diarrhea and bovine rotavirus, applied in the direction of recombinant DNA technology, microbial measurement/inspection, DNA/RNA fragments, etc. Less demanding, more specific effects

Active Publication Date: 2021-05-28
NORTHWEST A & F UNIV
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Problems solved by technology

[0006] At present, there are only reports on the application of RPA technology to detect a single type of bovine diarrhea virus, for example, Chinese patents CN104894118A and CN107974513A involving the detection of BVDV, and Chinese patent CN108546779A involving the detection of BRV, while the detection of BCoV h...

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  • Triple RPA (recombinase polymerase amplification) detection kit for bovine viral diarrhea virus, bovine coronavirus and bovine rotavirus
  • Triple RPA (recombinase polymerase amplification) detection kit for bovine viral diarrhea virus, bovine coronavirus and bovine rotavirus
  • Triple RPA (recombinase polymerase amplification) detection kit for bovine viral diarrhea virus, bovine coronavirus and bovine rotavirus

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Embodiment Construction

[0043] The present invention will be described in further detail below in conjunction with the accompanying drawings and embodiments. The examples are only used to explain the present invention, not to limit the protection scope of the present invention.

[0044] (1) Primer design and screening

[0045] (1) Collect BVDV genome sequences (57 pieces) in GeneBank, use MegAlign to compare and analyze the sequences, and select the 5'UTR region that is conserved within the species and specific among the species as the detection target gene of BVDV; select BCoV according to the literature data The RdRp gene and the VP6 gene of BRV are used as detection target genes, and the target gene sequences are further compared to determine the candidate region of the gene sequence to be amplified, and then primers are designed according to the determined gene sequence.

[0046] The following primer screening principles are adopted to eliminate the primers that do not meet the following require...

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Abstract

The invention discloses a triple RPA (recombinase polymerase amplification) detection kit for a bovine viral diarrhea virus, a bovine coronavirus and a bovine rotavirus. The triple RPA detection kit comprises RPA detection primer groups for the bovine viral diarrhea virus, the bovine coronavirus and the bovine rotavirus, wherein the RPA detection primer groups are respectively composed of nucleotide sequences shown as SEQ ID NO.1 and SEQ ID NO.5, SEQ ID NO.8 and SEQ ID NO.13, and SEQ ID NO.15 and SEQ ID NO.18. The kit is based on a recombinase polymerase amplification method, cDNA formed by RNA reverse transcription is used as a template for amplification reaction, and a detection result is obtained by nucleic acid gel electrophoresis. The kit can be used for simultaneously detecting the bovine viral diarrhea virus, the bovine coronavirus and the bovine rotavirus, is simple to operate, has relatively high sensitivity and specificity, and provides a rapid detection reagent for on-site screening of the bovine diarrhea virus.

Description

technical field [0001] The invention relates to the field of viral nucleic acid detection, in particular to a triple RPA detection primer set, kit and detection method for bovine viral diarrhea virus, bovine coronavirus and bovine rotavirus. Background technique [0002] Diarrhea is a common disease in cattle. There are many reasons for cattle diarrhea, among which viral infection is the main reason. With the expansion of cattle raising scale, the incidence of calf diarrhea is on the rise, and the damage is the most serious. As far as the current situation is concerned, the viruses that cause bovine diarrhea mainly include bovine viral diarrhea virus (BVDV), bovine coronavirus (BCoV) and bovine rotavirus (Bovine rotavirus, BRV). [0003] BVDV belongs to Flaviviridae and Pestivirus. Its genome is single-stranded positive-sense RNA. The natural hosts of BVDV include pigs, cattle, sheep and other ruminants. It can cause fever, mucosal erosion, diarrhea, and reproductive disor...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/6844C12N15/11
CPCC12Q1/701C12Q1/6844C12Q2600/16C12Q2521/507C12Q2522/101C12Q2537/143Y02A50/30
Inventor 赵晓民童德文王凯丽刘志豪
Owner NORTHWEST A & F UNIV
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