Establishment And Application Of One Step RT-PCR Detection Method For Porcine Japanese Encephalitis Virus

Porcine Japanese encephalitis is a vector mosquitoes spread by the Japanese encephalitis virus (Japanese encephalitis virus, JEV),is one kind of Zoonoses.JEV can cause the pig reproductive failure, causing huge economic losses to the swine industry in China.the study about the Japanese encephalitis virus one-step RT-PCR detection kit provide technological support and protection for farming industry in china.The main content of this study are as follows:Porcine Japanese encephalitis virus RNA extraction method of stuties:In order to find appropriate RNA extraction method for JEV three different RNA extraction methods(Guanidinum thiocyanate,Trizol reagent and RNA exaction kit)were compared and evaluated.UV and electrophoresis were employed to test the purity and integrity of extacted RNA.The result shows:The RNA which extacted by guanidinum thiocyanate has got the lowest purity and integrity.And the RNA which extracted by Trizol reagent and RNA extraction kit has got satisfactory purity and integrity.For the difference of extraction efficiency between RNA extraction kit and Trizol reagent is not significant,considering the cost,Trizol reagent is chosen experiment. consider convenient to select RNA extraction kit as the RNA extraction method.The test kit to extract the virus solution RNA diseased sample RNA was extracted using Trizol reagent.Establishment of One Step RT-PCR detection method for Porcine Japanese encephalitis:A pair of primers located at the conservative district of JEV SA-14-14-2gene was designed,and the specificity of the primers was also tested;One-step RT-PCR was established and optimized,determine the total reaction program:42℃30min,94℃3min,94℃30s57℃30s72℃40s30cycles,72℃5min;ultimately determine50ul reaction system::taqE0.5ul (2.0U), AMV reverse transcriptase enzyme0.5ul (2.0U), magnesium ions5ul (2.5mmol/L), ultrapure dNTP5ul, RT-PCR buffer5ul, Forward and Reverse of each primer1ul, ddH2O29ul. the sensitivity of the method can reach5pg.Through the repeatability and stability tests show the method has a satisfactory repeatability and stability; Japanese encephalitis virus (JEV), classical swine fever virus (CSFV),porcine reproductive and respiratory disorder syndrome virus (PRRSV) virus amplification, the results show that the reagent cartridge is highly specific for JEV.Preliminary application of porcine Japanese encephalitis virus One step RT-PCR detection method:The method was applied to detect8parts of JEV vaccine in miceJEV vaccine immunized mice,artificial infected mice and natural infected mice.The result shows:negative result was got when JEV vaccine immunized mice were detected;The method can detected artificial infected mice in early period; The9parts of detection result was the same with tradition detection methods.Positive samples and artificially infected samples were detected by one-step RT-PCR,two-step RT-PCR and ELISA method.the result shows:the kit is1.5h faster than two-step RT-PCR when detecting,and the cost is also savedThe result shows that:above the method, which is established and the optimized porcine Japanese encephalitis one-step RT-PCR method has a sensitive, reliable, specific and stable, and the advantages of convenient, quick, is suitable for clinical use, for Detecting our country in aquaculture JEV provides a practical technical method.