Real-time fluorogenic quantitative PCR detection kit for bovine viral diarrhea-diarrhoea virus and special primer and probe thereof

Abstract

The invention discloses a real-time fluorogenic quantitative PCR detection kit for type-I and type-II bovine viral diarrhea-diarrhoea virus and a special primer thereof and a TaqMan probe. The detection kit has advantages of simple operation, strong specificity, high sensitivity and good repeatability for BVDV detection. The detection kit can be used not only for identification of type-I and type-II bovine viral diarrhea-diarrhoea virus and hog cholera virus but also for accurate quantification of type-I and type-II bovine viral diarrhea-diarrhoea virus. The detection kit can be used in fields of detection of raw materials and semi-finished products in vaccine production and detection of dairy products, etc.

Description

technical field [0001] The invention belongs to the detection of veterinary animal pathogens in the technical field of biological detection, in particular to a real-time fluorescent quantitative PCR detection kit for qualitative and quantitative detection of type I and type II bovine viral diarrhea-mucosal disease viruses and its special purpose Primers and TaqMan probes. Background technique [0002] Bovine viral diarrhea - mucosal disease [0003] Bovine viral diarrhea-mucosal disease (BVD-MD), referred to as bovine viral diarrhea or mucosal disease, is caused by bovine viral diarrhea-mucosal disease virus (BVDV), and mainly occurs in cattle An acute, febrile infectious disease characterized clinically by mucous membrane inflammation, erosion, necrosis, diarrhea, and abortion in pregnant cows. Bulletin No. 96 of the Ministry of Agriculture of my country promulgated in 2008 listed bovine viral diarrhea disease into three types of diseases. [0004] Bovine viral diarrhea-...

AI Technical Summary

Problems solved by technology

[0014] At present, for the detection of BVDV virus, the methods commonly used in the industry include ordinary PCR detection technology, fluorescent quantitative PCR detection technology and RPA detection technology. Strong sex, leading to specific bands appearing in viruses with high nucleic acid sequence homology, resulting in false positives, and the sensitivity of RPA detection technology is lower than that of fluorescent quantitative PCR detection technology, such as the detection sensitivity of RPA patent document CN201510292386.2 2TCID only 50 , while the detection sensitivity of quantitative PCR patent document CN201410658803.6 is as high as 0.32TCID 50 , but due to the different positions of the primers and probe sequences designed by different fluorescent quantitative PCR methods, the sensitivity of BVDV virus detection is not nearly the same. The establishment of "the detection virus sensitivity in the literature is only 10 2 copies / uL, and the method was established only for type I BVDV virus and not for type II BVDV

Since the nucleotide sequences of type I and type II BVDV viruses are relatively different, it is very difficult to find a good site for the detection of type I and type II BVDV at the same time. Although the detection method established in patent document CN201410658803.6 has good repeatability, The highest standard deviation of the cycle number is only 0.6, but because the BVDV strain used in this patent is the C24V strain, which belongs to type I BVDV, and the literature does not involve type II BVDV strains, whether this method can detect type II BVDV BVDV strains need further confirmation

In addition, BVDV detection also faces the interference of classical swine fever virus (CSFV), because classical swine fever virus (CSFV) and BVDV are members of the same genus, have high homology, and have strong serological cross-reactivity. Diffusion tests, immunofluorescence techniques, enzyme-linked immunosorbent assays, virus neutralization tests, and conventional PCR techniques all have certain cross-reactions, and it is difficult to completely distinguish the two, so there are false positives in BVDV detection

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