The Development Of Visible LAMP Method For IBRV Detection And A Brief IBRV Epidemiological Investigation On Two Farms

Infectious bovine rhinotracheitis(IBR),also known as necrotizing rhinitis and red rhinopathy,causes morbidity of cattle including fever,dyspnea,rhinitis,inflammation of upper respiratory passage.The febrile and contagious disease is casued by infectious bovine rhinotracheitis virus(IBRV)which belongs to Bovine herpesvirus 1(BoHV-1),and is becoming a main cause of cattle industry loss.Nowadays,with large scale and intensive development of dairy and cattle industryand the increasing trade in genetic materials and livestock at home and abroad,the IBR is becoming more prevalent.Moreover,vaccines of IBR have not been supplied on the market yetand neitherhas the plan of monitoring and controlling been implemented in China.The majority of epidemiological investigation is about the detection of serum antibody currently.Available data indicates that the IBR has been spreading in China,and cattle industry has suffered heavy loss.Thus,it is of importance to carry out study on the pathogen of IBR.Usually the epidemiological investigation on IBRV can not be carried out on the site as a result of restriction of laboratory's condition.The aim of this study is to develop an on the site detection method to analyze the epidemic state of IBRV.The mothod was developed based on the loop-mediated isothermal amplification(LAMP)for fast detection.The research consists of two sections.Part 1:Establishement of rapid LAMP detection of IBRV judging by colour reactionIn order to establish the chromogenic LAMP detection method of IBRV,we designed and synthetised three pair of primers based on the conserved regions,then optimized the reaction system,and successfully established the LAMP method.The method can detect 10 copies of plasmids containing target sequence within 50 minutes and doesn't cross react with other virus,and the results can be judged by naked eyes directly.The detection results showed the serious infection of IBRV in dairy farms,the positive rates of sera and nasal swaps are 48.2%-69.4%and 79.4%-95.7%respectively with averages of 56.5%and 84.4%respectively.The establishment of this method brought convenience for IBRV detection on the site and laid the foundation for the development of rapid detection kit.Part 2:Epidemiological Investigation of IBRTo verify the prevalence of IBR in Chongming Island and to conduct an epidemiological survey and analysis on IBR,nested PCR was used to detect IBRV from nasal swaps and sera of two dairy farms respectively.The results indicated that the positive rate of IBRV is similar whether in nasal swaps or serums compared with the LAMP method and their coincidence rate is high.By McNemar Test,the difference of both methods was not significant,and it proved the reliability of LAMP method.Analysis on etiology showed the sequence BHV-1/SH309 amplified from samples has a high homology of more than 97.8%with referenced strains and located the same branch with Brazil strain IBR 6813(DQ006851),BH81(DQ006854),LD30/2(DQ006855)and LD15(AY7583820).The study provided a theoretical support to formulate suitable measures about prevention and control of IBRV.