The Establishment And Application Of BVDV QRT-PCR Detection Method And The Preliminary Establishment Of Fluorescent Microsphere Test Strips

Bovine Viral Diarrhea(BVD)is caused by bovine viral diarrhea mucosal disease virus(BVDV),and it is an endemic disease in cattle in most parts of the world and is considered one of the most important infectious agents in the livestock industry due to its high prevalence and sustained economic losses in dairy and cattle.BVDV spreads within and between groups.It can cause diarrhea,reproductive disorders,intestinal and respiratory diseases,and even cause persistent infections(PI),so reducing PI animals is the main cause of controlling clinical morbidity and economic losses caused by BVD.At present,the methods for detecting BVDV mainly include ELISA,test strips,etc.,but their cost is high in large-scale sample testing,so it is of great significance to establish an economical,simple,and rapid method.This study intends to establish BVDV SYBR Green II real-time quantitative PCR(QRT-PCR)and preliminary establishment of E0,E2 fusion protein fluorescent microsphere test strip detection method,and the main pathogen(BVDV)of dairy cow diarrhea in Heilongjiang region.According to the NS3 gene sequence of BVDV V027 strain in GenBank,three pairs of specific primers were designed for the conserved gene segment,and the target gene was amplified by RT-PCR.The target fragment was ligated into the cloning vector pMD-18T to construct a standard plasmid.It is a template,and the conditions of QRT-PCR are optimized on two instruments respectively,and the sensitivity of the two instruments is compared;And compare the established QRT-PCR method with the test results of the commercial kit;E0,E2 fusion protein was expressed and polyclonal antibody against rabbit fusion protein was prepared.After purification,it was diluted to different concentration gradients and coupled with fluorescent microspheres.A one-step fluorescent microsphere test strip of E0 and E2 fusion protein was initially established,and the repeatability,specificity and sensitivity of the test strip were detected.The results showed that the BVDV NS3 plasmid was successfully constructed,and the established BVDV QRT-PCR method has good specificity,sensitivity and stability.The coincidence rate of BVDV QRT-PCR and commercialization kit was 100%,and BVDV purification test was carried out on cattle in some areas of Heilongjiang.The results showed that the positive rate of field group was 75%,and the total detection rate of PI cattle was 0.33%;The established test strip has specificity and stability,and the antibody is diluted 10~3 times at most to bind well with BVDV.The qRT-PCR method based on BVDV NS3 gene has the characteristics of sensitivity,specificity and high reproducibility.It is suitable for different instruments,and the epidemic situation of BVDV antigen in our province is clarified.Finally the detection method of fluorescent microsphere test strips of BVDV E0 and E2 fusion proteins was preliminarily established.This experiment provides an effective method for large-scale and rapid detection of BVDV antigen,and provides theoretical support for the purification of BVDV in cattle farms.