Development And Application Of Pcr And Elisa Method For Detection Of Mycoplasma Suis

Mycoplasma suis (M. suis) is a kind of uncultivable bacteria that attach to the surface of host erythrocytes. This pathogen can cause infections which are clinically marked, either by an overt life-threatening haemolytic anaemia or a mild chronic anaemia, by illthrift, and infertility. The disease caused by M. suis widly spread in the world, leading to serious economic loss in the pig industry. However, not too much study on the diagnostic methods has been reported so far.16S rRNA sequence of M. suis was relatively conserved and used to classify different clinical isolates. The surface-localised protein MSG1is a candidate virulence factor of M. suis which involved in the adhesion to erythrocytes. The main contents of the research were as following:1. Estabilshment and comparison of the PCR and FQ-PCR assays for detection of Mycoplasma suisPCR and FQ-PCR methods were developed using primers and fluorescent-labeled TaqMan probe according to the nucleotides sequence of16S rRNA gene of M. suis. Plasmid containing16S rRNA gene was used as template to optimize the conditions of PCR and FQ-PCR reactions. The detection limits of PCR and FQ-PCR were104and10copies of nucleotide, respectively. The sensitivity of FQ-PCR was higher than PCR by1000times. But the results of detection of S. choleraesuis, ETEC,S. suis, P. multocida, M. hyopneumoniae, H. parasuis or PCV2were all negative.20clinical samples were detected by these two assays, positive rate was60%(12/20) by PCR, while75%(15/20) by FQ-PCR. It indicated that these two assays have good sensitivity and specificity.2. Preparation and identification of monoclonal antibodies against MSG1protein of Mycoplasma suisThe monoclonal antibodies (MAbs) were prepared by fusing mouse myloma cells (SP2/0) with spleen cells from BALB/c mice immuned with purified recombinant MSG1protein of M. suis. Two hybridoma cell lines secreting MAbs against MSG1protein were screened by indirect enzyme-linked immunosorbent assay (ELISA) and named as1A7and3G6, respectively. Western blot and indirect immunofluorescence assy (IFA) results showed that the two MAbs could react specifically with the recombinant MSG1as well as natural MSG1protein. ELISA titers in supernatant and ascites of these two MAbs were1:4096,1:1024and1:1638400,1:51200, respectively. Serial passage results confirmed that the titers of the MAbs were stable during20passages in vitro. The isotype of1A7and3G6both belong to IgGl/к and the two MAbs reacted with the same antigen determinant.3. Development of blocking ELISA for the detection of antibody to Mycoplasma suis by using peroxidase-labelled momoclonal antibodyA blocking ELISA method was developed based on a M. suis MSG1protein and its specific monoclonal antibody (MAb). The reaction conditions for each step were optimized: the coating antigen was0.5μg/mL; the sealing buffer was5%skim milk, and the sealing time was3h at37℃; sera samples diluted by1fold and incubated1.5h at37℃; MAb-HRP dilution is5000fold and incubated1h at37℃; The TMB substrate was added and incubated at37℃for15min before terminated with stop solution. The results of50M. suis negative serum samples were statistically analyzed, and the cutoff value of blocking ELISA was determined:samples presenting a percentage inhibition (PI) of≥37.25%were considered positive; samples with a calculated percentage inhibition of≤25.44%were rated negative and those presenting a blocking effect between25.44%and37.25%were considered doubtful. One hundred serum samples were tested by indirect ELISA and blocking ELISA, the results showed that the sensitivity and specificity of the blocking ELISA were92%and100%, respectively. In the repeatability test, both the intra-batch and inter-batches variation coefficients were lower than10%. Recombinant antigen had no cross reaction with the antibodies to PCV2, HPs, PRV, PRRSV, CSFV and EMCV. These results suggested that the established blocking ELISA is specific, sensitive and reproducible. This method was used to detect310clinical serum samples from different areas of China, resulting a positive rate of52.26%, which indicating that the M. suis wide spread in many swine farms.In summay, in this study, two MAbs against MSG1protein of M. suis were prepared, and the PCR and ELISA methods were successfully developed, which providing useful tools for disease diagnosis and epidemiological investigation of M. suis.