多重半套式聚合酶链反应在检测脑脊液病原菌中的应用

目的：建立多重半套式聚合酶链反应（PCR）快速检测脑脊液标本中常见的病原菌。方法：通过对病原菌16S rRNA基因保守区和变异区的序列分析，设计通过引物及革兰阴性菌、革兰阳性菌的特异性引物，分别作为外、内侧引物，对脑脊液标本中不同细菌的DNA进行多重半套式PCR扩增；同时与常规细菌培养法作比较，并检测了该方法的敏感性。结果：外侧扩增后革兰阳性菌、革兰阴性菌经扩增后均有长约1032bp的片段产生；内侧扩增两种细菌除1032bp的产物外，革兰阳性菌另有一336bp的特异性产物，革兰阴性菌另有一127bp的特异性产物。该方法最低可检测出8cfu/ml的大肠埃希菌；62份脑脊液标本扩增结果与培养法相比，敏感性、特异性、阳性预测值、阴性预没值分别为93．8％、5．7％、88．2％、97．8％。结论：多重半套式PCR方法能特异、敏感、快速地检测出脑脊液感染的常见病原菌。

Polymerase chain reaction of multiple half sleeve type application in the detection of cerebrospinal fluid pathogen

Objective To establish a new method of multiplex semi nested polymerase chain reaction (PCR) to detect pathogens in cerebrospinal fluid (CSF). Methods According to the analysis of the conservative and variable regions in bacterial 16S rRNA genes, we designed universal primers for all bacteria and specific primers for most gram positive and gram negative bacteria. All primers were added into the same reaction systems successively of a two step PCR assay to amplify the different bacterial DNA in CSF, and the results were compared with common culture method with sensitivity and the specificity both detected at the same time. Results Both gram positive and gram negative bacteria amplified DNA fragment about 1 032 bp after first step amplification with universal primers. In the second step, specific fragments of 336 bp and 127 bp were amplified in gram positive and gram negative bacteria respectively besides fragments of 1 032 bp ; The detection limit for E.coli was 8 cfu/ml. The comparison of 62 CSF samples decteced by both multiplex semi PCR and conventional culture method revealed sensitivity, specificity, positive and negative values of 93.8 %, 95.7 %, 88.2 %, and 97.8 % respectively for PCR. Conclusion The result suggested that the multiplex semi nested PCR we established was sensitive, specific and rapid method for clinical laboratory to detect pathogens in CSF.