Establishment Of The Multiplex PCR For Bovine Parainfluenza Virus 3 And Other Relative Pathogenic Microorganism

To establish the PCR detection method for Bovine parainfluenza virus 3(BPIV3)and other relative pathogenic microorganism,we designed three pairs of specific PCR primers according to the hemagglutinin-neuraminidase(HN)protein genomic sequences of three BPIV3 genotypes obtained from Gen Bank,and four pairs of specific PCR primers based on 5'UTR?gC?opp D/F,HN protein genomic sequences for M.bovis?BVDV?IBRV ?BPIV3.Meanwhile,the primers ratio and annealing temperature were specifically optimized.Finally,two multiplex PCR(mPCR)detection method was established successively.The results showed that the proposed three genotypes-based multiplex RT-PCR has high sensitivity and specificity on detecting BPIV3,and it can effectively be applied in clinical sample detection.Meanwhile,the proposed Quadruple PCR also get high sensitivity and specificity for the detection of BPIV3,M.bovis,BVDV,IBRV.Compared with traditional single PCR,which using 45 samples,the proposed mPCR get the same detection result.The experiments showed that the proposed mPCR has high effectivity and sensitivity in detecting the four pathogenic microorganisms at the same time.To investigate the epidemiology of bovine parainfluenza virus type 3(BPIV3)infections in four farms,a serological survey was conducted by ELISA method for 88 serum samples targeting at BPIV3 antibody.The results showed that the samples collected from all of the four surveyed farms were positive for the BPIV3 antibody.Among the four farms,the highest positive rate was 100%(22/22),the lowest positive rate was 86.36%(19/22),and the average positive rate was 95.45%(84/88).