Development Of Immunological Diagnostic Methods And Nanogold Nucleotides Diagnostic Method To PPRV

Peste des petits ruminants (PPR), is a higly contagious viral disease affecting domestic and wild small ruminants. In recent years, it spread to new areas in the world. PPR was firstly outbroken in Tibet of China in July 2007. It becomes more necessary and critical for us to reinforce studies on diagnosis and control of the disease.Using the plasmid which contains the complete PPRV N gene and five pairs of designed primers according to the conservatism of N gene, the five subunits were amplified by PCR. Then the subunits were cloned into pET-32a (+) expression vector successfully. The subunits protein were purified.The results by Western blot showed that four of the subunits of N protein had immunoreactivity with PPRV antibodies, only one did not have. The results by ELISA showed that the main B cell epitopes sited on the fifth subunit, in other words sited on IV zone.The fifth subunit protein pET-PPRV-N5 which had strong immunoreactivity and high specificity was purified and used as coating antigen to developed a indirect ELISA to detect the antibodies against PPRV. The established indirect ELISA was sensitive and specific. Compared with standard competitive ELISA, the coincidence rate between two methods was 98.4%.To develop a gold immunochromatographic assay (GICA) for detection of PPRV antigen, polyclone antibody was purified from hyper-immuneserum and conjugated with colloidal gold particles prepared by the tri-sodium citrate reduction method. Meanwhile, purified recombinant PPRV N protein and the anti-goat IgG were striped in test line position and in the control line position respectively. The minimum antigen detected by developed GICA was 10 ng. The results showed that the GICA was specific and easy to perform. It was an acceptable alternative for the use in field diagnosis.To detect micro amount of PPRV nucleotide molecules, a gold nanoparticle-based method with silver staining enhancement was established. A pair of probes which were complementary to target nucleotides were designed and labeled with biotins and nanopaticles respectively. Then two kinds of probes were hybridized with target nucleotides, the hybridization products were immobilized to microplate via avidin-biotin system, and the signals of gold nanoparticles were amplified with silver staining solution. This method could detect as few as 1 pmol/L of target PPRV nuclotides.In general, three methods were established to detect the antibody, antigen and nucleotides of PPRV. These methods would be useful in the detecting of PPRV.