The Establishment Of Fluorescent Quantitative PCR Assay Detection Method For Infectious Bovine Rhinotracheitis Virus And Isolation And Identification Of IBRV NM8 Strain

Infectious bovine rhinotracheitis(IBR),also known as"red nose disease"or"necrotizing rhinitis",is a highly contagious disease caused by infectious bovine rhinotracheitis virus,namely bovine herpesvirus type I(BHV-1).The natural host of IBRV is cattle,which can infect the respiratory system,reproductive system and nervous system,causes huge economic losses to the global cattle industry.Most countries adopt vaccine immunization measures to control IBRV infection.Among them,the use of gene deletion vaccine can distinguish vaccine immunization and wild virus infection,which provides an effective means for the eradication of IBRV.In order to understand the epidemic situation of IBRV,two real-time fluorescent quantitative PCR method for detection of infectious bovine rhinotracheitis virus were established.The detection method was established by designing specific primers for the conserved regions of IBRV TK and g B genes and optimizing the reaction conditions.The results showed that the detection methods for TK and g B genes had good specificity,repeatability and sensitivity;the two real-time fluorescence quantitative PCR detection methods established have the same annealing temperature and can be carried out at the same time,which will greatly save experimental time;In addition,the two real-time fluorescence quantitative PCR detection methods which could be used to distinguish the infection of IBRV wild virus and TK gene deleted vaccine strains.Two established methods were used to detect 219 clinical samples.The positive rates were 18.72%and17.81%respectively,while the positive rate of conventional PCR was 15.98%.It showed that the two established detection methods were more sensitive than conventional PCR.In order to understand the prevalence of IBRV,two Real-Time Fluorescent Quantitative PCR methods for detection of infectious bovine rhinotracheitis virus were established.The detection methods were established by designing specific primers for conserved regions of IBRV TK and GB genes respectively,and optimizing the reaction conditions with the principle of the minimum CT value and the maximum fluorescence intensity increment.If the two were not consistent,the CT value will be given priority.The results showed that the Real-Time Fluorescent Quantitative PCR methods for TK and GB genes had good specificity;The lowest detection limit was 7.80×10~1 copies/?L and 5.47×10~2 copies/?L;The intra assay coefficients of variation ranged from0.10%to 0.71%and from 0.09%to 0.33%,and the inter assay coefficients of variation ranged from 0.03%to 3.39%and from 0.21%to 1.28%,indicating good reproducibility.The annealing temperature of the two Real-Time Fluorescent Quantitative PCR detection methods were 55?and they can be carried out at the same time,which will greatly save the experimental time.Meanwhile,the two methods can be used to distinguish the infection of wild virus and TK gene deleted vaccine strains,which will provide technical support for the detection of IBRV.219clinical samples were detected by the two methods and conventional PCR.The positive rates were18.72%,17.81%and 15.98%respectively,which indicated that the two methods were more sensitive than conventional PCR.In this study,g C,TK,g G,g D and g E gene sequences of IBRV NM8 strain were obtained by PCR amplification.Nucleotide homology analysis,amino acid homology analysis,multiple sequence alignment and phylogenetic analysis of 5 genes were carried out.The result of homology analysis showed that the IBRV NM8 strain had the highest homology with the BHV-1.1 reference strain,followed by the BHV-1.2 reference strain,and had the lowest homology with the BHV-5reference strain.The results of multiple sequence alignment showed that there were 1,1,2 and 2amino acid variations in the g C,TK,g G and g E genes,respectively.There was no variation in amino acids deduced from the g D gene.No amino acid insertion or deletion was found in the five genes,which indicated that the NM8 strain was well conserved.The results of genetic evolution analysis showed that the g C,TK,g G,g D and g E genes were located in the same evolutionary branch as the BHV-1.1 reference strains,and different branches from the BHV-1.2 and the BHV-5 reference strainsIn summary,this study provides new data for further study of IBRV in China,and provides new information for epidemiological investigation of IBRV.It will provide new virus strains for the development of new IBRV detection technology and vaccine in China in the future.