猪肺炎支原体检测芯片的研制及初步应用  

Development and application of microarray for detecting Mycoplasma hyopneumoniae

作　　者：肖国生[1] 曹三杰[1] 黄小波[1] 文心田[1] 

机构地区：[1]四川农业大学动物医学学院基因芯片实验室/动物疫病与人类健康四川省重点实验室

出　　处：《中国预防兽医学报》2008年第1期57-63,共7页Chinese Journal of Preventive Veterinary Medicine

基　　金：教育部长江学者和创新团队发展计划基金资助项目(IRT0555-9);四川省生物技术重点项目(01NG018-01);重庆三峡学院引进人才科研资助项目资助(2006-SXXYRC-09)

摘　　要：本研究探索建立一种新的基因芯片方法检测和鉴定猪肺炎支原体。在猪肺炎支原体的16SrRNA、P36和P463个基因内设计3个靶基因片段,用PCR标记Cy3探针,建立了用于检测和鉴定猪肺炎支原体的检测芯片。试验结果显示,猪肺炎支原体与检测芯片的3个靶基因(Mhp-16S、Mhp-P46和Mhp-P36)杂交呈阳性,猪鼻支原体只与Mhp-16S靶基因杂交呈阳性,其它所测细菌和病毒不与检测芯片的靶基因杂交。检测芯片的检测灵敏度和PCR相同,能达到10pg基因组DNA/50μL标记体系或反应体系。用检测芯片鉴定了3株疑似猪肺炎支原体临床分离株,结果有2株为猪肺炎支原体,1株为其它猪支原体。用检测芯片、P36-PCR和分离鉴定分别对45头病猪临床样品选择混合培养物中的猪肺炎支原体进行了检测,结果它们的检出率分别为20%(9/45)、22.2%(10/45)和8.9%(4/45)。检测芯片还检出有26.7%(12/45)的病猪感染了其它猪支原体。试验结果表明,所建立的检测芯片是一种快速检测和鉴定猪肺炎支原体的敏感特异性方法。A new microarray-based assay was developed for detecting and identifying M.hyopneumoniae using Cy3-labelled probes and 16S rRNA, P36, and P46 as target genes. The results showed that probes of M.hyopneumoniae could hybridize with Mhp-16S, Mhp-P46, and Mhp-P36 genes, but probes of Mycoplasma hyorhinis could only hybridiz with Mhp-16S gene. The micr-oarray assay had a sensitivity of detecting 10 pg of M.hyopneumoniae DNA, which was same as PCR method. Forty five clinical samples from pigs were tested for M.hyopneumoniae by different methods, of which 20 % （9/45） were detected positive by microarray, 22.2 % （10/45） by P36-PCR and 8.9 % （4/45） by M.hyopneumoniae isolation. In addition, the microarray also detected 26.7 % （12/45） positive for other Mycoplasma. The results indicated that the detecting microarray is a highly sensitive assay for identification and detection of M.hyopneumoniae.

关 键 词：猪肺炎支原体 基因芯片 鉴定 

分 类 号：S852.62[农业科学—基础兽医学]