Development And Application Of TaqMan Real-time PCR Assay For Detection And Quantitation Of Japanese Encephalitis Virus

Japanese encephalitis (JE) caused by Japanese encephalitis virus (JEV) is a mosquito-borne disease, which threatens the health of humans and animals. The disease is prevalent in China, the Southeast Asia, India and the Western Pacific Region, and spread continuously throughout the world. Not only cause serious damage to human health, but also lead loss to the swine industry. Therefore, it is urgent to develop a rapid and accurate method for the detection and control of the disease.Traditional virus isolation methods such as intracranial inoculation assay in suckling mice and the BHK-21cell isolation and identification method take a long time; The sensitivity of the Latex agglutination test is low; ELISA is not suitable for early diagnosis; The conventional PCR is only qualitative and need other operations; Therefore, it is essential for us to develop a rapid and sensitive method for JEV early diagnosis. Compared with the above methods, the real-time PCR assay has many advantages such as rapid, sensitive, specific and quantitative.In this study, a TaqMan real-time PCR was developed for detection of JEV with a pair of primers targeting the E gene of the virus, yielding amplicon with size of1500bp. Link the target fragment with pMD-18T, then the recombinant plasmid pMD18-T-JEV-E was constructed. Serial diluted plasmids ranging from108copies/?L to101copies/?L were used as standards. The PCR conditions, including the concentrations of primers and probe and the procedure were optimized to obtain optimal specific fluorescent signals. The correlation coefficient was0.996and the PCR efficiency was99.4%. The test results showed that the assay was specific for JEV detection and no crossing-reaction with other related porcine virus, such as encephalomyocarditis virus, classical swine fever virus, porcine reproductive and respiratory syndrome virus, pseudorabies virus, porcine circovirus and porcine parvovirus. The assay could detect a limit of10copies/?L of plasmid templates, which was100times more sensitive than the conventional PCR. The repeatability tests showed that the inter-and intra-variation were both less than2%. The real-time PCR assay had the same sensitivity with that of intracranial inoculation assay in suckling mice, and more sensitive than the BHK-21cell isolation and identification method. The viral loads of the viscera in mice were also detected by the method. The established assay could be potentially used for detection of JEV in clinical samples and the evaluation of vaccine. It is of great importance to study the pathogenicity of JEV.