TaqMan-MGB探针法快速检测脑膜炎奈瑟菌的研究

TagMan-MGB Probe Real-time Fluorescence Quantitative PCR for Rapid Detection of Neisseria meningitidis

目的建立TaqMan-MGB探针荧光定量PCR快速检测脑膜炎奈瑟菌(Nm)方法,为流脑的诊断提供更加灵敏、特异、可标准化、自动化的检测方法;评价TaqMan-MGB探针荧光定量PCR法在健康人群带菌调查中的应用价值。方法根据Nm ctrA基因的3端保守序列设计合成引物和TaqMan-MGB探针,建立快速检测脑膜炎奈瑟菌的荧光定量PCR方法,并对反应条件进行优化。结果TaqMan-MGB探针荧光定量PCR检测脑膜炎奈瑟菌的灵敏度为102cfu/ml,TaqMan-MGB探针法检测900份健康人群咽拭子标本,阳性34例,检出率3.8%,高于培养法(1.2%)。结论该方法特异性强、灵敏度高,能实现脑膜炎奈瑟菌的快速检测,适用于健康带菌检查并可作为临床常规诊断方法的补充,有利于流脑的早诊断、早治疗。

Objective To establish a TaqMan-MGB probe real-time fluorescence quantitative PCR, which would be specific, sensitive, standardized, and automated, for rapid detection of Neisseria meningitidis directly from clinical samples. To evaluate the application of the method in surveying the situation of taking Neisseria menin- g/t/d/s carrier in healthy persons. Methods Primers and TaqMan-MGB probes were designed and synthesized according to the high conservative sequence of target gene ctrA of Neisseria meningitidis in Genbank, and then real-time fluorescence quantitative PCR for detection of Neisseria meningitidis. Results The TaqMan-MGB probe real-time PCR was specific, and the detection limit for Neisseria meningitidis was 10^2cfu/mL. 3.8% （34/900） throat swabs samples Were positive by TaqMan-MGB probe real-time PCR. The results indicated that the real-time PCR was more sensitive than cultivation method （ 1.2% ）. Condution The TaqMan-MGB probe real-time PCR established in this study could detect Neisseria rneningitidis from samples in 3h. It could be applied for surveying the situation of Neisseria meningitidis carrier in healthy persons and was beneficial supplement of traditional methods, which would contribute to the early diagnosis and freatment of epidemic cerebrospinal meningitis.