猪肺炎支原体量子点荧光原位杂交研究方法的建立与应用  

Development and application of quantum dots fluorescence in situ hybridization for detection of Mycoplasma hyopneumoniae

作　　者：王占伟[1] 熊祺琰[1] 冯志新[1] 刘茂军[1] 路璐[1] 范志鹏[1] 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兽用生物制品工程技术重点实验室国家兽用生物制品工程技术研究中心,江苏南京210014

出　　处：《中国兽医学报》2012年第12期1832-1836,共5页Chinese Journal of Veterinary Science

基　　金：国家自然科学基金资助项目(31001080);国家农业科技成果转化基金(2009GB2C100129);江苏省农业科技自主创新基金资助项目(cx(09)619);中国博士后科学基金面上资助项目(20090461075)

摘　　要：利用生物素化的猪肺炎支原体核酸探针和量子点荧光显色系统,对人工感染组、自然感染组和健康对照组的试验猪肺样品,以及临床样品进行量子点荧光原位杂交检测。结果表明:按照探针质量浓度为1 mg/L、80～90℃变性10 min、37℃杂交10 h和QDs-SA复合物浓度为10 nmol/L的条件进行猪肺炎支原体的量子点荧光原位杂交检测结果比较理想;而人工感染组、自然感染组和健康对照组样品的QD-FISH检测结果与分离培养结果和PCR检测结果相比,符合率均达到100%;临床样品的QD-FISH检测结果与PCR检测结果相比,符合率为83.3%。从而证明猪肺炎支原体量子点荧光原位杂交法是一种直观和敏感的检测方法,可以用于猪肺炎支原体的检测、定位和致病机理研究,也为其他病原的研究提供了参考方法。CISH was performed by using a biotinylated DNA probe specific for P36 gene of Mhp. For visualization, Quantum dots（QDs） as chromogen was used. Lung tissue samples collected from experimentally infected pigs,naturally infected pigs, healthy pigs as negative control and suspected infected pigs were detected. The results showed that the detection effect of Mhp was ideal by QD-FISH in accordance with the conditions of the probe concentration t mg/L,denaturation 10 min at 80-90 ℃ ,hybridization 10 h at 37 ℃ and QDs-SA hybridization complex concentration 10 nmol/L. Moreover,the result was 100% compliance by comparing with QD-FISH and PCR for artificial infection group,natural infection group and control group, and 83.3% compliance for clinical samples. This research provides an intuitive and sensitive CISH method to detect Mhp which would help to study the in vivo location of Mhp and the pathogenic mechanism of Mhp infection. Moreover,this technique would also provide a method for studing other pathogens.

关 键 词：猪肺炎支原体 量子点 量子点荧光原位杂交 斑点杂交 检测 定位 

分 类 号：R852.62[医药卫生—航空、航天与航海医学]