Establishment And Application Of Fluorescence Quantitative PCR Detection For Porcine Reproductive And Respiratory Syndrome

Porcine reproductive and respiratory syndrome (PRRS) is an econmically important disease of swine, estimated to cost approximately US$ 560 million per year. in USA The syndrome is caused by an RNA virus referred to as PRRS virus (PRRSV), which is classified in the farily artericiride. Swine macrophages are the only indigenou cell type known to support PRRSV replication. 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, fluorescence quantitative PCR for detecting PRRSV was developed and simultaneously studied the proliferation axiom in the marc-145 cells and in vivo and distribution, and provides the basis for PRRSV epidemiology, disease prevention related research. The main contents are as follows:1. A pair of specific primers were designed,the expected fragment was amplificatied from RNA of test samples,The purified PCR product was connected with pGEM-T-easy vector and then transferred into JM109.The standard recombinant plasmid was gained form positive bacterium clone, As template and then reaction parameters were optimized to develop a real-time quantitative PCR assay. The results showed that there is a good linear relationship between the Ct value and the concentration gradient of standard plasmid cDNA specimen,and more reproducibility and specificity than traditional PCR.Three samples were examined using the real-time PCR repeatedly, and the results indicated that the real-time quantitative PCR was more reproducibility than traditional PCR and could be used for the diagnosis of PRRSV infection.2. Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) could grow in the Marc-145 cell line, but the proliferation characterization is still unknown. This study was investigated the replication disciplinarian of PRRSV in the Marc-145 line using Fluorescence Quantitative PCR. The results showed that the virus began to replicate obviously at the 12 h after inoculation. The viral proliferation was increased in a great speed between 18 and 36 h and reached the peak at 48 h after inoculation. This study provides basic information for investigating the pathogenic mechanism and vaccine production for PRRSV.3. Fluorescence quantitative PCR(FQ-PCR)assay was developed for rapid detection of PRRSV.Thirty five-day old pigs were artificially infected PRRSV strain.The distribution of the virus in the porcine body was detected by FQ-PCR.The results were as follows:At hour 12 post-infection(PI),the PRRSV could be detected from blood,heart and liver.The PRRSV reached peak quantity at 48 h PI,and could be detected from a variety of organs.The peak persisted up to 96 h PI and then declined quickly.At 168 h PI,however,the PRRSV could only be detected from feces,liver and spleen.This study provides a foundation for research of pathogenic mechanism of PRRSV infection.