猪肺炎支原体套式PCR检测方法的建立及应用  

Establishment of A Nested PCR Assay for Detection of Mycoplasma hyopneumoniae

作　　者：逯晓敏[1,2] 冯志新[1] 刘茂军[1] 吴叙苏[1] 甘源[1] 张映[2] 邵国青[1] 

机构地区：[1]江苏省农业科学院兽医研究所农业部动物疫病诊断与免疫重点开放实验室国家兽用生物制品工程技术研究中心,江苏南京210014 [2]山西农业大学动物科技学院,山西太谷030801

出　　处：《江苏农业学报》2010年第1期91-95,共5页Jiangsu Journal of Agricultural Sciences

基　　金：公益性行业(农业)科研专项(200803020);中国博士后基金项目(20070421022);江苏省农业科技自主创新基金项目[CX(08)608]

摘　　要：根据GenBank中登录的猪肺炎支原体P36(L-乳酸脱氢酶)全基因序列,设计合成了2对引物,建立套式PCR方法。运用该方法对猪肺炎支原体不同菌株培养物进行了扩增,并对经肺内免疫猪支原体肺炎活疫苗后支气管肺泡灌洗液进行了检测。结果表明,不同菌种均能扩增出427 bp的目的条带,而其他病原菌未出现特异性扩增条带。建立的套式PCR方法检测猪肺炎支原体的最低检测限度为10个CCU(颜色变化单位)。支气管肺泡灌洗液检测结果表明,经肺内免疫的猪支气管肺泡灌洗液扩增结果均为阳性。Two pairs of primers were designed according to the sequence of P36 （L-lactate dehydrogenase） gene of Mycoplasma hyopneumoniae published in GenBank, and a nested PCR assay was established for the detection of M. hyopneu- moniae. Different M. hyopneumoniae strains and the bronchoalveolar lavage fluid of intrapulmonic immunization with attenua- ted M. hyopneumoniae vaccine were detected with nested PCR. A specific 427 bp fragment was amplified from different M. hyopneumoniae strains by the developed nested PCR. Non-M. hyopneumoniae did not show specific amplified fragment. The nested PCR had high sensitivity to for detection of 10 CCU M. hyopneumoniae. The results of the bronchoalveolar lavage fluid detected by nested PCR showed that the amplified results was positive by intrapulmonic immunization.

关 键 词：猪肺炎支原体 P36 套式PCR 

分 类 号：S858.282.62[农业科学—临床兽医学]