多重半套式PCR检测细菌16srRNA基因方法的建立

（1）目的 设计并建立多重半套式聚合酶链反应（PCR）快速检测临床标本中感染病原菌DNA方法。（2）方法 通过对多数病原菌16srRNA基因保守区和变异区序列分析，设计通用引物和革兰阴性菌及革兰阳性菌的特异性引物分别作为外侧和内侧引物，分两步先后加入同一反应体系中对不同细菌的DNA进行扩增。（3）结果金黄色葡萄球菌和大肠无纪律希菌经扩增后均有长度为1032bp的DNA片段产生；金黄色葡萄球菌另有一长度为336bp的特异性产物，大肠埃希菌另有一长度为127bp的特异性产物。（4）结论 该方法可以特异、敏感、快速检测出临床感染的病原菌。

ESTABLISHMENT OF AN ASSAY TO DETECT BACTERIAL 16s rRNA GENES WITH MUTIPLEX SEMI-NESTED PCR METHOD

Objective\ To design and establish a new method to dectect pathogenic bacteria DNA in clinical infectious samples by mutiplex semi nested PCR. \ Methods\ According to the analysis of conservative and variable regions in bacterial 16s rRNA genes ,we designed universal primers for all bacteria and specific primers for gram positive and gram negative bacteria. All primers were added successively into the same reaction system of a two step PCR assay to amplify the different bacterial DNA. Results\ Both E.coli and S.aureus amplified a 1 032bp DNA fragment. In addtion,specific fragments of 336bp and 127bp were amplified in S.aureus and E.coli respectively . \ Conclusion\ The result suggests that the multiplex semi nested PCR we established is a more sensitive,specific and rapid method for a clinical laboratroy to detect bacterial pathogenes.