Diagnosis Establishment Formulation Of RT-PCR,Fluorescence Quantitative PCR And Mutiplex RT-PCR Detection For Japanese Encephalitis Virus

Japanese Encephalitis Virus (JEV) causes reproductive failure in swine, manifested as embryonic resorption, fetal mummification, abortion and stillbirths. The virus is ubiquitous among swine throughout the world and is enzootic in most herds that have been tested. In this research, the RT-PCR method and fluorescence quantitative RT-PCR for the detection of JEV was developed.1. A pair of primers was synthesized from the Japanese Encephalitis Virus cDNA sequences published in GenBank. Through optimizing RT-PCR conditions, the expected 349 bp fragment was amplified from cDNA of JEV-infected PK-15 cells, The amplified fragment was shown to be specific for JEV cDNA after digested by EcoRâ… . This method could detect the template cDNA of 10fg at least. These results showed the RT-PCR technique is a fast, simple, economical, specific and sentitive detection method.2. A pair of primers and the corresponding TaqMan probe were designed, the expected fragment was amplified from RNA of JEV-infected PK-15 cells, The purified PCR product was connected with pGEM-T-easy vector and then transferred into JM109.The standard recombinant plasmid was gained from positive bacterium clone.The plasmid PCR and plasmid sequence mensuration showed that the expected fragment was successfully cloned. Series of diluted standard recombinant plasmid cDNA specimen were amplified by real-time quantitative PCR, which indicate that there is a good linear function in statistics between the Ct value and the concentration gradient of standard plasmid cDNA specimen. Analysis of the dynamic curve, under this condition of the reaction, the sensitive degree is 90 copies. The construction of real-time quantitative PCR provides the basis for the early and rapid detection and analysising the infect degree of JEV .3. On the basis of single RT-PCR for JEV and SIV established, by optimizing action conditions, The Multiplex RT-PCR was developed. The results showed that two specific fragments of 349 bp for JEV and 155 bp for SIV could be amplified in the multiple RT-PCR simultaneously. DNA fragments were not amplified from cultural cells challenged with PCV-2 and control cultural cells (PK-15), This method could detect the template cDNA of 100pg for JEV and 10pg for SIV, This method could differentiate JEV, SIV and mixed infection.4. On the basis of single RT-PCR for JEV,SIV and PRRSV established, by optimizing action conditions, The Multiplex RT-PCR was developed. The results showed that two specific fragments of 349 bp for JEV,155 bp for SIV and 426 bp for PRRSV could be amplified in the multiple RT-PCR simultaneously. DNA fragments were not amplified from cultural cells challenged with PCV-2 and control cultural cells (PK-15), This method could detect the template cDNA of 100pg for JEV,10pg for SIV and 1000pg for PRRSV, This method could differentiate JEV, SIV, PRRSV and mixed infection.