Bovine Respiratory Disease Virus Serological Survey And Multiple RT-PCR Diagnostic Method Establishment And Preliminary Application

There are various bovine respiratory tract virus disease, also the corresponding pathogenic are variety. This research mainly aims at the bovine respiratory syncytial virus, bovine parainfluenza virus type3virus and bovine viral diarrhea virus. Bovine respiratory syncytial virus (BRSV) belong to the Paramyxoviridae, Pneumovirinae, pneumovirus, single strand of negative strand RNA virus, is one of the main pathogens of respiratory system disease of calves, with worldwide distribution, resulted in greater economic losses to the cattle industry. Bovine parainfluenza virus type3(BPIV3) is the Paramyxoviridae, paramyxovirus member, single strand of negative strand envelope RNA virus. BPIV3can make cows suffering from bronchitis and pneumonia, often leading to secondary infection of cattle industry, great harm, industry resulted in greater economic losses to the cattle. Bovine viral diarrhea virus (BVDV), also known as mucosal disease virus (MDV) or bovine viral diarrhea mucosal disease virus (Bovine Viral Diarrhea-Mucosal Disease, BVD-MD), belonging to the Flaviviridae, the pestivirus, single stranded positive strand RNA virus. BVDV infection of cattle, the clinical symptoms may manifest as mucosal disease, diarrhea, female abortion, stillbirth and fetal malformation, caused great economic losses to the cattle industry.This research includes two parts, the first part is the bovine respiratory syncytial virus, bovine parainfluenza virus type3and bovine viral diarrhea virus serological epidemiology. Collected803sera from Xinjiang, used neutralization to test sera, the number of bovine respiratory syncytial virus antibody positive serum was435, the positive rate was54.17%; the number of bovine parainfluenza virus type3antibody positive serum was271, the positive rate was33.75%; the number of bovine viral diarrhea virus antibody positive serum was523, the positive rate was65.13%.The second part is establish the three triple RT-PCR method of detecting bovine respiratory syncytial virus, bovine parainfluenza virus type3and bovine viral diarrhea virus. According to the bovine respiratory syncytial virus, bovine parainfluenza virus type3and bovine viral diarrhea virus gene sequences published on the Gen Bank, respectively, design primers in the3conservative regional for detecting virus. First, establish3kinds of single RT-PCR detection method, on the basis, optimization of multiple RT-PCR reaction conditions, established3virus three RT-PCR technology, amplification of bovine respiratory syncytial virus was287bp, bovine parainfluenza virus type3was506bp, bovine viral diarrhea virus was975bp specificity fragment. Establishment of multiplex RT-PCR method for the detection of BRSV, BPIV3and BVDV sensitivity is achieved respectively32pg/L,164pg/L,21pg/L, very sensitive. With the establishment of multiplex RT-PCR method for the detection of BRSV, BVDV, BPIV3, IBRV, MDBK cell freezing medium, the results were consistent with the expected. Using the multiplex RT-PCR detection method to detect positive samples, virus and the negative control3times, identical results. The78clinical materiasl were detected by three RT-PCR method and the single RT-PCR method, the results was both the total coincidence rate in90%above. This indicates that the three RT-PCR detection method has the advantages of convenient, quick, high specificity, and can be used for the3viruses at the same time detection and differential diagnosis.To carry out this experiment has important significance. Through the experimental research, can be used for prevention of BRSV, BPIV3and BVDV communication, popular, reduce these three kinds of disease on Chinese cattle industry losses.