PCR-微孔板核酸杂交技术检测肺炎衣原体研究

Detection of Chlamydia Pneumoniae (CPn)by Polymerase Chain Reaction and Microplate Hybridization Technique

建立一种新的PCR ELISA技术模式 ,简便、特异、敏感地检测肺炎衣原体 ,为肺炎衣原体的检测提供一种有效的新方法。采用链霉亲和素包被于聚乙烯微孔板 ,生物素标记的引物扩增目的DNA片段与荧光素标记的探针在微孔板内进行分子杂交 ,抗荧光素标记的辣根过氧化物酶可以与之结合 ,再加入底物即可产生颜色反应 ,通过测定其吸光度来进行结果判断。新建立的PCR ELISA方法与其它相关的六种病原体无交叉反应 ,临床标本检测检出率高 ,呼吸道感染者标本 42份检出率达 2 1 95 % ,肺炎患者标本 465份检出率为 42 1 5 % ,比套式PCR方法及其它检测方法的检出率都高。应用新建立的PCR ELISA方法检测肺炎衣原体特异、敏感 ,结果客观、稳定、可靠 ,对肺炎衣原体引起的疾病的诊断有十分重要的意义。

To establish a sensitive, simple and specific diagnostic technique for detecting CPn,an effective technique(PCR-ELISA )was developed. The streptavidin was coated in the microplate,the CPn DNA was amplified with the primers labeled by biotin,the probe was labeled by fluorescence.The PCR production and the probe was hybridized in the microplate, becoming conjugate after added the anti-FITC-POD,substrate(TMB and H 2O 2)made the conjugate emerged color reaction. Result was expressed in absorbency measured on a microplate reader. In specific test only CPn DNA was positive,the six related other pathogens were negative.Positive rates of detecting 42 respiratory tract infections specimens were 21 95%,465 pneumonia patients were 42 15%.The positve rate was higher than other techniques. PCR-ELISA is a sensitive,specific,objective and reliable one of detecting CPn DNA, it is possessing important significant in diagnosis diseases caused by CPn.;