Development Of An NASBA-ELIAS For Detection Of Highly Pathogenicporcine Reproductive And Respiratory Syndrome Virus

Porcine Reproductive and Respiratory Syndrome (PRRS) was one of infectious disease, which characterized by severe reproductive failure in swine, respiratory diseases and high mortality in piglets. The study aimed in establishing one detection method of high sensitive, specific, convenient and suitable for basic application, named nucleic acid sequence based amplification.Three specific primers were designed to amplify the Nsp2genes of FJ07A strains, The T-vector cloning strategy was employed to generate the recombinant plasmid for sequencing. The Nsp2gene sequences were analysed using DNAStar and compared with other PRRSV strains. The results showed that the homologous of FJ07A strains nuclear acid was29.1%with LV and98.5%-99.1%with HUN4strains、JXwn06strains、Henan-1strains、BJSY-1strains, Compared with classical American PRRSV strains, the homologous of FJ07A strains nuclear acid was81.2%.Sequence analysis of the Nsp2genes by phylogenetic trees indicated that the FJ07A strains was American mutants.The NASBA was developed for the amplification of the Nsp2genes of FJ07A strains, followed by a liquid phase hybridization of biotin-streptavidin in micro plates and enzyme labeled color reaction to detect the amplified products, then through the optimal reaction conditions and finally established the NASBA-ELISA method to detect the high mortality PRRS. The specific test of ELISA confirmed that this method only amplified the RNA of FJ07A strains; The sensitive test of ELISA suggested that this method could detect the NASBA amplified products with the dilution of5.2pg/μl.The results showed that the NASBA-ELISA can detect high pathogenic PRRSV sensitively and specifically,and suitable for detecting the PRRSV in clinic.