山羊传染性胸膜肺炎病原快速诊断方法的建立  

Establishment of Rapid Diagnosis on Pathogens of CCPP

作　　者：张双翔[1] 周碧君[1,2] 程振涛[1,2] 文明[1,2] 王开功[1,2] 崔亚兰[1] 李泽民[1] 覃岚[1] 钟文彬[1] 

机构地区：[1]贵州大学动物科学学院,贵阳550025 [2]贵州省动物疫病研究室,贵阳550025

出　　处：《生物技术通报》2012年第3期95-99,共5页Biotechnology Bulletin

基　　金：贵州省科学技术基金[黔科合J字(2011)2332号];贵州大学引进人才科研项目[贵大人基合字(2010)35号];贵州省科学技术基金项目[黔科合J字(2010)2108号];贵州省农业厅兽医科技计划项目(201006)

摘　　要：为准确快速鉴定临床疑似山羊传染性胸膜肺炎(CCPP)病原,针对丝状支原体山羊亚种(Mmc)与绵羊肺炎支原体(Mo)16S rRNA基因设计两对特异性引物Ps/Pa、Ms/Ma,通过优化反应条件及体系,建立检测CCPP两种主要致病支原体的双重PCR方法,并检测了所建方法特异性、敏感性及临床应用。当引物、Mg2+及dNTP浓度分别为0.8 pmol/μL、1.5 nmol/μL、0.2 nmol/μL,Ps/Pa与Ms/Mo比例为1 1时,于54℃退火40 s,可最小检出Mmc与Mo核酸量分别为0.1 ng、0.01 ng,敏感性良好。通过特异性与临床疑似病例检测,证明所建方法具较好特异性,可初步应用于临床诊断。To identify the pathogens of suspect CCPP rapidly and accurately,two special primers Ps/Pa and Ms/Ma which aimed 16S rRNA gene of Mmc and Mo were designed.The duplex-PCR method to detect two pathogens of CCPP was established by optimizing annealing temperature,amplification system and primer proportion.The results showed that effect of method was improved when the concentration of primer,Mg2＋ and dNTP reached 0.8 pmol/μL,1.5 nmol/μL,0.2 nmol/μL,equal mixed primers and annealed 40 s at 54℃.It could detect the minimum nuclear weight of Mmc and Mo was 0.1 ng,0.01 ng,which showed proper sensitivity.It was also proved satisfactory specificity and could be used in clinical diagnosis preliminarily.

关 键 词：山羊传染性胸膜肺炎 病原 双重PCR 建立 

分 类 号：S858.27[农业科学—临床兽医学]