Establishment Of A Multiplex PCR Method For Bovine Viral Respiratory Disease And Analysis Of IBRV Gene Sequences

Bovine respiratory disease complex（BRDC）is a worldwide multifactorial infectious disease and a major factor that endangers the cattle breeding industry in China.IBRV,BCo V,BRSV,and BPIV3 are the main viruses causing BRDC,however,the clinical symptoms caused by these agents are extremely similar and clinical diagnosis is a challenge,therefore,pathogenetic diagnosis is particularly significant in differentiating these agents.Based on the characteristics of these four pathogens,IBRV,BCo V,BRSV and BPIV3,we selected the conserved genes of IBRV g B gene,BCo V N gene,BRSV F gene and BPIV3 HN gene,designed specific primers,and established a quadruple PCR assay for IBRV,BCo V,BRSV and BPIV3 with amplified fragment sizes of 185bp,597bp and 260bp,respectively,597bp,260bp and 400bp,respectively.In this thesis,we firstly conducted a preliminary exploration of single-weight PCR methods,and the lowest detection lower limits of the four pathogen single-weight PCR methods for IBRV,BRSV,BPIV3,and BCo V were 3.72×10^-7 ng/μL,3.14×10^-6 ng/μL,2.86×10^-7 ng/μL,and 2.92×10^-5 ng/μL,respectively.By optimizing the reaction system and reaction procedure of the multiplex PCR method,the annealing temperature,extension time,number of cycles,primer volume and enzyme content of the one-step multiplex PCR were 60℃,30s,30 times,0.6μL and 1.6μL,respectively.the minimum detection limits for IBRV,BRSV,BCo V and BPIV3 were 3.72×10^-3ng/μL,3.14×10^-6ng/μL and 3.14×10^-6ng/μL,respectively,The detection limits were 3.14×10^-6ng/μL,2.86×10^-4ng/μL,and 2.92×10^-6ng/μL for IBRV,BRSV,BCo V,and BPIV3,respectively.96 clinical samples were tested,and the positive compliance rate was 96.3%compared with the classical single-duplex PCR assay.The IBRV material clinically detected in a region of Hebei was isolated and subjected to second-generation whole gene sequencing,and the experimental isolate was nearest related to Australian strain B589 after phylogenetic tree analysis.The genetic sequences of HBKS-IBRV segregates g B,g D,g C,and g E were also characterized for their homologies and evolutionary tree construction and the structural functions of the protein sequences by several online analysis facilities.These data provide valuable reference materials for the molecular epidemiology of IBRV strains,physicochemical characterization of major functional proteins,and also lay the foundation for future studies related to genetic variation of the virus.The results showed that the g B,g D,g C and g E proteins of HBKS-IBRV isolates were hydrophilic proteins withα-helix primary structure and 7,3,3 and 2 N-glycosylation sites and 14,7,6 and 5 ubiquitination sites,respectively.g B protein did not contain secretory signal peptide,while g D,g C and g E proteins contained secretory signal peptide;The g B and g D proteins contain two transmembrane structural domains,and the g C and g E proteins contain one transmembrane structural domain.