Establishment And Application Of Loop-mediated Isothermal Amplificationmethod In Detection Of The Bovine Herpes Virus Type 1

Bovine herpesvirus type 1 (BHV-1), also known as Infectious Bovine Rhinotracheitis (IBRV), is one of the serious pathogen on the cattle industry, the disease is prevalent in the world. Normal cattle will get a series of adverse symptoms after infection by this virus, such as dyspnea, respiratory and tracheal mucosa inflammation, etc. Meanwhile it can also cause female and male calf meningitis and reproductive system inflammation. In particular, the virus can lurk in a tissue of the body after infection, which and lead to persistent infection and long-term or even life-long poison, which may cause a huge impact and loss to the entire herd. At present, the infection and epide-mic of the disease were reported in the whole country. There is no relevant vaccine and diagnostic reagents in the domestic market.Because there is no commercial vaccine, in order to control the disease, monitoring the persistent infection and purifying the cattle were performed in the production process. While the traditional virus isolation technology, PCR technology and immunofluorescence detection technology were restricted to applicate in the field because of the time consuming, high cost or requiring high accurate instruments. The loop mediated isothermal amplifycation (LAMP) has the advantages of high sensitivity, high specificity, simple operation, low equipment requirement and the result evaluation is simple, rapid and suitable for the diagnosis level.In this study, according to the published relevant literature, the conserved region of BHV-1 was selected base on the comparison of the whole genome sequence in Genebank, three pairs of specific LAMP primers were designed by Explorer Primer design software. LAMP standardized detection procedu-res for BHV-1 had been established by the optimization of the amplification temperature, primer concentration, amplification time, betaine concentration.Finally LAMP detection technology for BHV-1 will be evaluated by studying the specificity, sensitivity and visibility of the system. Furthermore,46 cattle nasal swab samples were collected from cattle which suffered from respiratory diseases and detected by established LAMP detection technology. Comparing with the traditional PCR detection method and virus isolation method, we found that the detection rate (19/43) of LAMP detection method was significantly higher than that of the traditional PCR method (9/43), and similar to virus isolation method (17/49) after 3 generation. It shows that the LAMP detection method established in this study has high practical appli-cation value, and has the advantages of less time-consuming, short detection time. According to the booming cattle market and the lack of detection techn-ology, the LAMP detection method will have a very broad application pro-spect.