Establishment And Primary Application Of BVDV,BPV And BPIV Probe QRT-PCR Detection Methods

With the improvement of productivity,the country has made great efforts to develop animal husbandry.Dairy cattle and beef cattle breeding industries are gradually becoming large-scale,mechanized and industrialized.Formerly,some infectious diseases which generally are the sending out show a trend of outbreak.Under such circumstances,the monitoring pathogen is put into the high level.Nowadays,the frequent import and export trade breakes the local limitation and accelerates the spread of these diseases.Additionally,some diseases are subclinical infections,which have become the most potential dangerous factors of livestock production and veterinary medicine.Therefore,effective technology of diagnosis and treatments are an essential part of livestock trade quarantine in present.In this study,the real-time quantitative PCR(qRT-PCR)was applied to establish three TaqMan probe qRT-PCR and a duplex TaqMan qRT-PCR,respectively.After comparing a number of sequences of BVDV type 1 5'-UTR genes,BPV major structural protein VP2 genes and BPIV nucleoprotein N genes in GenBank,three pairs of specific primers and probes with different fluorescence groups were designed to establish single TaqMan qRT-PCR for BVDV,BPV and BPIV,and a duplex qRT-PCR for simultaneous detection of BPV and BPIV was also established.Through optimizing the reaction conditions suchu as annealing temperature,primer concentration and probe concentration,the results showed that the established probe qRT-PCR and duplex TaqMan qRT-PCR both had good repeatability and sensitivity.The specificity of BVDV qRT-PCR,and the method could only amplification BVDV,but not virus of the same family and genus or virus of the same family.Standar plasmid pMD18-T-BVDV 5'-UTR was detected for repeatability test and the correlation coefficient was more than 0.999%.With highly sensitivity,the detection limits was 1.55copies/?L in ordinary PCR 100000-fold by BVDV probe qRT-PCR.The reproducibility was better with intra-assay and inter-assay Ct values both less than 1.7%.Similarly,the established detection methods of BPV or BPIV probe qRT-PCR also had highly specificity,sensitivity and repeatability.The detection limits of BPV and BPIV were 10,000 times and 100 times as much as ordinary PCR,respectively,and there was no amplification for other pathogens.The intra-assay and inter-assay Ct values was both less than 1.65%.43 clinical samples were detected by BPV qRT-PCR.These results showed that 4 samples were positive for BPV.On the basis of better specificity,sensitivity and repeatability,the establishment of duplex fluorescence quantitative PCR can reduce the wast of reagents and samples,save time cost and improve work efficiency.When BPV and BPIV duplex qRT-PCR were used to detect common bovine pathogens,only the cDNA of BPV and BPIV could be specifically amplified,indicating that the method had better specificity and the probes did not interfere with each other.The method was able to detect2.0×10~1copies/?L pMD18-T-BPV and 2.0×10~2copies/?L pMD18-T-BPIV,and the coefficient of variation was both less than 1.2%in the intra-assay and inter-assay repeatability tests.