羊肺炎支原體內(nèi)蒙古分離株的同源性研究及雙重PCR檢測方法的建立.pdf

1、分類號 S852 學(xué)校代碼 10129 U D C 11.220 學(xué) 號 2009201107 羊肺炎支原體內(nèi)蒙古分離株的同源性研究及雙重PCR 檢測方法的建立 Studies on Homology of Mycoplasma ovipneumoniae from In

2、ner Mongolia Isolated Strains and Construction of Duplex PCR Determine Assay 申 請 人：張明月 學(xué)科門類：農(nóng) 學(xué) 學(xué)科專業(yè)：臨床獸醫(yī)學(xué) 研究方向：反芻動物抗病性研究 指導(dǎo)教師：吳樹清 教授 論文提交日期：二〇一二年五月Studies on Homology of Mycoplasma ovipneumoniae from Inner Mongolia Is

3、olated Strains and Construction of Duplex PCR Determine Assay Abstract Mycoplasma pneumoniae of Sheep and Goat is a kind of infectious disease with highly infectiosity caused by several mycoplasmas , it has become one of

4、 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

5、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 My

6、coplasma are Mycoplasma ovipneumoniae, confirming Mycoplasma ovipneumoniae can infect both sheep and goats. We also extract DNA of mycoplasma, then amplificat, cloning objective fragment and determine 16SrRNA sequence. c

7、ompared with Mycoplasma ovipneumoniae international mode strain Y-98 . analysis results show the two isolates and the mode strain possess homology of 99.8%. RAPD technique used for matching difference on genome between t

8、he 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 strain

9、s 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 on 16SrRNA

10、 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 t

11、ime, and use this method for testing clinical samples. The result shows: mode strains Y-98 and PG3 amplificated the 200bp and 400bp fragment consistent with the expected results, two laboratory isolates and Y-98 strain s

12、howed the same result. The control group display the negative and had good specificity; sensitive experiments show that the method can detect 5pg Mmc DNA and 0.05pg MO DNA or detect 1ng Mmc DNA and 1ng MO mixture,with a