Development And Application Of DNA Microarray For Diagnosing The Subtypes Of JEV

Japanese encephalitis (JE), which is caused by JE virus (JEV), is one the most important viral encephalitis in the world Japanese encephalitis virus (JEV) belongs to the family flaviviridae and is transmitted between animals and human host by culex mosquitoes. Japanese encephalitis is prevalent through-out Eastern and Southern Asia and the Pacific Rim. The majority of JE victims are children and nearly half of the surviving patients have cognitive or motor sequelae. Purified formalin inactivated mouse brain derived vaccine and live attenuated vaccines (SA14-14-2) are available; the latter is reported to be safe, effective and cheap. The role of chimeric recombinant attenuated JE vaccine is under investigation. In the absence of specific antiviral therapy,, JE is managed by symptomatic and supportive therapies and preventive measures. So the early diagnosis of JEV is very important It is Laboratory diagnosis of JE is by IgM capture ELISA, Besides, the agar diffusion reaction, enzyme linked immunosorbent assay and other serological methods can identify JEV. Whereas these methods are all lower sensitivtive and hadro-dependence by experience.Although the molecular biology method can improve the sensitivty, it only test one subtype once, therefore it can not satisfy the need of detection. This study constructs DNA microarray for diagnosing the subtypes of JEV based on the diversity of PrMand E gene, which can diagnose the subtypes of JEV and the JEV at the same time. Major studies are processed as follows:1Design primer and probes:This part includes construct recombinant plasmid and design primer and probes.The conserved fragments were amplified by RT-PCR, and each fragment was cloned in vectors to construct recombinant plasmid respectively. After sequencing, the result was tested by using DNAStar and compared with the corresponding subetypes.Aliment reports show that the sequence is above94%with the bulk corresponding subetypes. And design primer and probes by using BLAST.2Optimizing the condition of hybridization:This part includes prepare the DNA microarray,probe concentration options, hybridization temperature and hybridization time options.The probes are diluted to30uM,and marked slide,37℃fixed to12hours,determined the preparation of genechip and condition of hybridization.The results show that the probe is40μM; hybridization temperature is42℃, the hybridization time is3hours.3Application of asymmetric PCR in DNA microarray:This part includes sensitivity, specificity, reproducibility testing and the asymmetric PCR rate. This study, there are four different titres were chose:1:25,1:50,1:100and1:200. It shows that these DNA microarray have specificity and good reproducitivity, and the sensitivity higher than PCR. the optimization primer concentration is1:50, The hybridization signal has significant enhance comparing with the conventional PCR.