多重聚合酶链反应快速检测脑膜炎中的病原体

Detection of meningitis pathogens by multiple polymerase chain reaction

目的 建立脑膜炎多重 PCR检测系统 ,以在一次扩增中快速、特异地同时检出新型隐球菌、结核杆菌、脑膜炎双球菌 ,用于脑膜炎病原体感染的快速诊断 .方法 根据新型隐球菌 URA保守序列、脑膜炎双球菌基因组中特定的 H.8外膜蛋白基因序列、结核分枝杆菌基因组中的 IS986插入基因序列片段 ,分别设计出 3对特异性寡聚核苷酸引物 ,采用多重PCR技术 ,同时检出脑脊液中的新型隐球菌、结核杆菌、脑膜炎双球菌 .结果 应用多重 PCR反应体系 ,对引物的相关性实验结果表明引物之间不会因相互干扰而出现假阳性结果 ;多重 PCR扩增的预期结果为 :单一菌感染出现一条特异性扩增区带 ;混合感染应出现 2条或 3条特异性扩增区带 ,经实验达到预期的扩增结果 ;对 15份已明确诊断的脑脊液标本 ,分别采用常规 PCR和多重 PCR进行同步扩增 ,结果两种扩增方法均能扩增出预期的目的片段 ,符合率达 10 0 % .对 2 0例临床脑脊液标本的 PCR扩增结果分别为 :新型隐球菌 15 % (3/2 0 )、结核杆菌 2 5 % (5 / 2 0 )、脑膜炎双球菌 10 % (2 / 2 0 ) .结论 多重 PCR有效地为临床病原体的混合感染 ,以及原因不明的脑膜炎患者的快速诊断 ,提供了快速、准确的诊断手段 .有效地减少了脑膜炎的误诊和漏诊 。

AIM To establish a multiple polymerase chainreaction (PCR) system to detect cryptococcus neoformans (CN), meningoccus and tuberculosis (TB) rapidly and specially for meningitis diagnosis. METHODS According to URA constant sequence of cryptococcus neoformans, H.8 extramembrane protein gene of meningoccus and IS986 insert gene sequence of tuberculosis, three pairs of specific oligonucleotide primers were designed and used in one multiple PCR detection system to detect the three pathogens at the same time. RESULTS The primers relativity assay showed there was no interference among them. The expectant result of multiple PCR was that one strip was amplified when it was a single pathogen infection and two or three special strips when it is a mixed infection. Our test proved it. Normal PCR and multiple PCR were used to detect 15 CSF samples of definitely diagnosed patients in the same conditions. The results of the two methods were accordant. The PCR results of the 20 clinical CSF samples were as follows: The positive rate of cryptococcus neoformans was 15% (3/20), meningoccus 25% (5/20) and TB 10% (2/20). CONCLUSION Multiple PCR is an effective method to diagnose mixed infection or uncertain meningitis rapidly and accurately. It can effectively decrease the faulty diagnosis of meningitis.