Studies On Homology Of Mycoplasma Ovipneumoniae From Inner Mongolia Isolated Strains And Construction Of Duplex PCR Determine Assay

Mycoplasma pneumoniae of Sheep and Goat is a kind of infectious disease with highly infectiosity caused by several mycoplasmas, it has become one of the most harmful communicable to the healthy development of our region’s sheep industry.In this study, Suspected Mycoplasma pneumonia of sheep and goat was isolated by clinical cases samples collected from the different regions of Inner Mongolia Autonomous Region. After the experiments of isolation and culture, morphology observing, biochemical tests and molecular biological methods to determine that the two isolates of the Mycoplasma are Mycoplasma ovipneumoniae, confirming Mycoplasma ovipneumoniae can infect both sheep and goats. We also extract DNA of mycoplasma, then amplificat, cloning objective fragment and determine16SrRNA sequence. compared with Mycoplasma ovipneumoniae international mode strain Y-98. analysis results show the two isolates and the mode strain possess homology of99.8%. RAPD technique used for matching difference on genome between the isolates and mode strain, The results showed that the DNA template of different strains had a significant difference in the electrophoresis strip size under the same primer and condition, illustrating the local strains of mycoplasma may exist some variation, and laid the foundation for the future to select representative strains for vaccine and drug research.The test also designed two pairs of specific primers based on16SrRNA sequence of the filamentous Mycoplasma capricolum subspecies and Mycoplasma ovipneumoniae, the Duplex-PCR detection method established can make the accurate differential diagnosis on two types of pathogens at the same time, and use this method for testing clinical samples. The result shows:mode strains Y-98and PG3amplificated the200bp and400bp fragment consistent with the expected results, two laboratory isolates and Y-98strain showed the same result. The control group display the negative and had good specificity; sensitive experiments show that the method can detect5pg Mmc DNA and0.05pg MO DNA or detect Ing Mmc DNA and Ing MO mixture,with a good sensitivity.