Development And Primary Application Of Pcr-Elisa Method For The Detection Of Mycoplasma Suis

Mycoplasma suis is a kind of prokaryotic pathogens which parasitize surface of red blood cell or in the plasma and marrow, it can cause clinical symptoms such as anemia, jaundice and fever. Mycoplasma suis can be spread through blood and other ways, serious damage to the animal husbandry and human health. There’s no potent drugs developed for this pathogens at present, so detection and grasp the infection status as early as possible is very important for the disease control. In this study, a PCR-ELISA method with high specificity, sensitivity, security and rapidly was established for detect Mycoplasma suis.A pair of conventional primers was designed according to the Mycoplasma suis OxaA membrane protein gene sequences which published in GenBank. The targeted gene fragment (Ms232) was amplified by using PCR techniques, then it’s cloned into the eukaryotic cloning vector pMD18-T to construct the pMD18-T-Ms232recombinant plasmids. The recombinant plasmids were identified by restriction endonucleases digestion and PCR, then the sequences of the cdna were obtained, the targeted gene fragment was identified. Refer to the sequences of conventional primers, the labelled primers were designed and it was labelled by Digoxin and Biotin. Amplified the targeted gene fragment by the labelled primers, got the targeted fragment which with Digoxin and Bictin. Coated ELISA plates with Streptavidin and added the targeted fragment, then it was caught by the biotin-streptavidin linkage. Then added the alkaline phosphatase marked anti-digoxin antibodies, it would combine with Digoxin which labeled on the fragment. Finally, added the corresponding chromogenic substrate and detected the OD405nm values. The PCR cycles, Streptavidin concentration, block buffer condition and colour-developing time were selected by improve labeling method, explore PCR plateau and square titration.The results shows that conventional primers could amplifie the Mycoplasma suis genetic fragments accurately, therefore the effectiveness of the labelled primers were ensured. In this study, the detected threshold was0.36pg of Mycoplasma suis DNA, the sensitivity was ten times as much as that of conventional PCR assay. And the PCR-ELISA could not detecte the DNA from Streptococcus suis, Mycoplasma haemocanis, Mycoplasma hyopneumoniae, Echerichia coli and health pig blood. Compared with conventional PCR-ELISA assay, the cost time was shortened nearly two hours. Seventy three DNA samples were detected by PCR-ELISA and PCR, the results showed that the positive rates were42.5%and34.2%.In summary, a PCR-ELISA assay for Mycoplasma suis with sensitivity, specificity, security and rapidly was established in this study, for the detection of M.suis. It provided a foundation for the early diagnosis and epidemiological investigation of Mycoplasma suis.