Comparison Of Four Tests Of Porcine Reproductive And Respiratory Syndrome Virus

Porcine reproductive and respiratory syndrome virus(PRRSV) is the causative agent of porcine reproductive and respiratory syndrome(PRRS). PRRS is considered one of the important infectious diseases of swine. The disease is associated with reproductive failure in sows and respiratory problems in young pigs. PRRSV can be diveded into the European(EU) type and the North American(US) type and the North American type is prevalent in China. The aim of this study was to establish four etiological diagnosis methods for North American type PRRSV and the characteristics of these methods were analyzed.Many studies have shown that PRRSV N protein is the most abdundant viral proteins and the most immunogenic protein.Pigs infected with PRRSV produces a strong immune response against the N protein. In this study, ELISA and indirect immunofluorescence assay (IFA) were established for detecting North American PRRSV using anti-N protein polyclonal antibody. In the ELISA,the concentration of coating virus was0.093μg/μL, and the optimal dilution of polyclonal antibody N was1/20and the concentration of HRP labled SPA was1/4000. In the IFA assay, the concentration of coating virus was300TCID50cytotoxic, the optimal dilution of the polyclonal antibody N was1/400and the concentration of the FITC labled SPA was1/400. Nevertheless, the accuracy and detection rates of the two assays were not very ideal.RT-PCR primers and RT-LAMP primers were designed according to the sequences of the NSP2gene of North American type PPRSV, RT-PCR and RT-LAMP were established to detect this virus. By detecting serial dilution of positive control, the RT-LAMP method was found to be103-104fold times more sensitive than conventional RT-PCR. To examine the detection limit, the serial diluted RNA of PRRSV HH08and JILINTN1were detected.The RT-LAMP assay was proved to be able to detect7.9×10-11μg/μL (JILINTN1) and8.3×10-10μg/μL(HH08) of virus RNA. The RT-PCR assay was proved to be able to detect virus7.9×10-6μg/μL(JILINTN1) and8.3×10-7μg/μL (HH08) of virus RNA. Both assays distinguished PRRSV from other control viruses. To test if the methods worked for clinical samples,40samples were detected.Among40samples,5samples were found positive in the RT-LAMP assay and5samples were found positive in RT-PCR assay. These results were consistent with virus isolation. This result is same with the results of virus isolation and RT-PCR universal primers amplified result. As accuracy and detection rates of the both methods were ideal,they can be useful candidate assays for detection of PRRSV.