The Establishment And Application Of NASBA-ELISA For Differential Diagnosis Of Porcine Reproductive And Respiratory Syndrome Virus Classical And Variation Subtypes

According to the published nucleotide sequences of porcine reproductive and respiratory syndrome virus (PPRSV) from GenBank, three methods of nucleic acid sequence-based amplification (NASBA) -ELISA (A), NASBA-ELISA (C) and NASBA-ELISA (V) for detection of American genotype PRRSV, and differential diagnosis of PPRSV classical strains and variant strains were developed in this study. They, together with PRRSV RT-PCR kit, were used to the detection of samples of pigs inoculated with different PRRSV strains, to compare the sensitivity of NASBA-ELISA and RT-PCR detecting PRRSV.Three groups of primers and probes were designed and synthesized according to the consensus sequences of PRRSV ORF7 genes, Nsp2 genes of ORF1a and Nsp2 genes deletion information. Three methods of NASBA-A, NASBA-C and NASBA-V which respectively amplify RNA of American genotype PRRSV, PPRSV classical strains and PPRSV variant strains were established by optimizing reaction conditions. NASBA products was analysed by agarose gel electrophoresis showed that the specific RNA fragment was amplified. The specificity of NASBA amplification was further verified by northern blot which also show that the detection probe was specific. And then ELISA to detect NASBA amplification products was established through the optimization of reaction conditions. It was showed that NASBA-ELISA (A), NASBA-ELISA (C) and NASBA-ELISA (V) have good sensitivity, specificity and reproducibility.Different age pigs were inoculated with different PRRSV strains. Blood and nasal swab samples were collected on a regular basis. Organs were collected following dead or euthanasia. All samples had been detected with NASBA-ELISA and RT-PCR kits. The results of NABSA-ELISA and RT-PCR coincided in 83.91% (219/261), NASBA-ELISA (C) and NASBA-ELISA (V) could accurately distinguish PPRSV classical strains and variant strains. The positive samples of blood, nasal swab and tissue in NASBA-ELISA were more than that in RT-PCR. And the results indicate that NASBA-ELISA is more sensitive than RT-PCR. Blood was more suitable than nasal swabs as samples for NASBA-ELISA.