| Bovine viral diarrhea virus(BVDV),infectious bovine rhinotracheitis virus(IBRV)and Akabanevirus(AKAV)are the main viral infectious diseases that can cause reproductive disorders in cattle and are widely spread worldwide.All three viruses can cause reproductive abnormalities in cattle,seriously threatening the healthy development of the cattle industry worldwide and bringing great challenges to the prevention and control of B VD,IBR and AKA in various countries around the world.Therefore,in this study,the following main contents were studied for the three pathogens:1.Serological investigation of BVDV,IBRV and AKAV in some areas of NingxiaIn this study,competitive ELISA was used to detect BVDV,IBRV and AKAV antibodies in clinical serum samples from four areas of Ningxia.The results showed that the positive rates of the three pathogenic antibodies were 89.13%,86.96%and 1.63%,respectively.The highest antibody positive rate of BVDV was 89.62%in Yinchuan area,89.05%and 1.63%in Wuzhong area,92.86%in winter,85.71%and 84.61%in spring and autumn,and 3.81%in autumn.2.Establishment and application of multiplex PCR assayIn this study,three pairs of specific primers were designed for the 5’-UTR of BVDV,the gB gene of IBRV and the S gene of AKAV,respectively.A multiplex PCR method was constructed on the basis of establishing and optimizing a single PCR for each pathogen,and the reaction system and conditions were optimized,and the specificity and sensitivity of this method were verified.The results showed that the constructed multiplex PCR method did not amplify other bovine-derived viruses,and the lowest detection limits for BVDV,IBRV and AKAV were 103copies/μL,103 copies/μL and 104copies/μL,respectively.The positive rates of BVDV,IBRV and AKAV were 18.48%,14.13%and 1.63%,the positive rates of BVDV and IBRV were 3.8%,and the positive rates of BVDV and AKAV were 1.63%.The coincidence rate of BVDV,IBRV and AKAV was more than 92%,and the coincidence rate of BVDV+IBRV and BVDV+AKAV was 100%.The results showed that the multiplex PCR assay established in this study could be used for the detection of clinical samples.3.Establishment and application of multiplex TaqMan fluorescent quantitative PCR assayPublished conserved sequences of BVDV,IBRV and AKAV were selected from GenBank.Three pairs of specific primers and probes were designed by online Primer-BLAST for BVDV 5’UTR,IBRV gB gene and AKAV S gene,respectively.Complementarity analysis was performed between each primer using MEGA-X software to preliminarily establish TaqMan single fluorescent quantitative PCR for each pathogen and optimize its reaction conditions.On this basis,a TaqMan multiplex quantitative fluorescence PCR assay was successfully constructed by a series of optimization of TaqMan multiplex quantitative fluorescence PCR reaction system and conditions,and it was verified that the specificity of this assay was good,and the lowest detection limits for BVDV,IBRV,and AKAV were 103 copies/μL,104 copies/μL,and 103 copies/μL,respectively.The coefficients of variation for the repeatability test were less than 4%for both intra-and inter-group.The positive rates of BVDV,IBRV and AKAV were 20.65%,15.22%and 1.63%,3.8%and 1.63%,respectively.In summary,this study found that BVD and IBR have been commonly infected in some areas ofNingxia by serological investigation,and also found that AKA infection has occurred in Wuzhong area of Ningxia,and the three viruses have different infection conditions in different regions and seasons.This investigation provides a theoretical basis for the comprehensive prevention and control of BVDV,IBRV and AKAV in cattle herds in Ningxia.In addition,multiplex PCR and multiplex TaqMan fluorescent quantitative PCR for BVDV,IBRV and AKAV were successfully established,which provided a quick and simple molecular biological detection method for the detection and epidemiological investigation of BVD,IBR and AKA in cattle herds in China. |
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