Establishment Of Indirect-ELISA, Real-time PCR And LAMP For Detection Of Porcine Circovirus Type 2

PCV2 (Porcine circorus type 2) is a member of the family circoviridae which was first encountered in 1974, and in the following years, it was shown that PCV2 was associated with many swine diseases, especially to the PMWS (Postweaning Multisysterm Wasting Syndrome). A report has documented the association of PCV2 with abortion, reproduction disorder and PRRS (Porcine Reproductive and Respiratory Syndrome). Available data indicates high seroprevalence of antibodies to PCV2 worldwide and it has caused excessive lost in global swine industry.In this study ,we establish three diagnosis methods to detect PCV2 DNA and serum antibody quickly, sensitively and exactly, including Isndirect ELISA, Real-time PCR and LAMP.1. The establishment of Indirect ELISA method for PCV2 detection.We use the prokaryotic expression system to get the His-Cap recombinant protein, and purified it with the Ni+affinity chromatography column.After SDS-PAGE electrophoresis and western blotting assay, the recombinant protein is 40Ku as designed and identified by special antibody to the PCV2.Then we choose the optimal His-Cap concentration and anti-serum dilution by block titration.Also the blocking reagent, blocking time, dilutions of HRP-labeled Ab, et al.were selected.In the inner and between assay, testing repeatability is good.Then we use the kit to test 60 samples of pig serum, menwhile taking the Porcine circorus type 2 ELISA testing kit from Corp.Shi Ji Yuan Heng as control.The coincidence between them is 91.7%.All the results suggested the Indirect ELISA assay a potentially useful tool for PCV2 detection.2. The establishment of Real-time PCR method for PCV2 detection.A pair of specified primers were designed according to the published sequence of PCV2(ORF2 gene) listed in GenBank.Meanwhile, the standard plasmid pGEX-6P-1-ORF2 containing sequence of ORF2 was constructed as the template.The optimization was carried out for Real-time PCR assay to detect the PCV2 quantitation. The results showed the sensitivity to PCV2 was 3.5×103Copies/μL, and the linear equation was expressed as Ct=-3.638×lg(Copies/μL)+41.399. No corss-reaction was found in other familiar viruses that can make pigs sick.The Real-time PCR assay effectively avoids contamination as regular-PCR.In research application and diagnosis of PCV2, the assay has potential value in use.3. The establishment of LAMP method for PCV2 detection.The conserved ORF2 gene was stiil picked for the LAMP assay as the target sequence. One set of primers were designed using PrimerExplorer4.0 software depending on some important parameters including: GC content, 5'-, 3'-term stability and secondary structures, et al.Two important parameters of LAMP were optimized including Mg2+ and outer and inner primers, Mg2+ and inner primers were found to influent most on amplification efficiency after research. The results showed the sensitivity to PCV2 was 9.5×102Copies/μL.No corss-reaction was found in other familiar viruses that can make pigs sick..Because LAMP method is rapid and easy to use, the established PCV2 LAMP is suitable for PCV2 detection, especially for screening large number of samples in farms.