TaqMan荧光定量PCR检测和鉴别不同血清群脑膜炎奈瑟菌方法的建立及应用

Establishment and applieation of TaqMan Real-Time PCR in detection and serogrouping of Neisseria meningitidis

目的建立TaqMan荧光定量PCR检测方法，用于脑膜炎奈瑟菌不同血清群菌株的检测和鉴别。方法设计合成7对引物和TaqMan探针，脑膜炎奈瑟菌种属特异性的基因为ctrA；不同血清群的脑膜炎奈瑟菌特异性基因分别为A群（sacB）、B群（siaD）、C群（siaD）、X群（xcbB）、Y群（synF）、W135群（synG）。检测和确定不同探针和引物用于脑膜炎奈瑟菌荧光定量PCR检测的特异性、敏感性，并同时将荧光定量PCR和乳胶凝集方法应用于121份疑似脑膜炎奈瑟菌感染患者的脑脊液标本的检测。结果ctrA、sacB、siaD（B群）、siaD（C群）、xcbB、synF、synG等7对引物和探针能准确检测和鉴定79株不同血清群的脑膜炎奈瑟菌菌株，检测灵敏性比相应的普通PCR高10^2～10^3倍，每对引物和探针反应体系中，能检测的最低全基因组DNA拷贝数分别为8、8、80、8、8、80、8；检测121份疑似脑膜炎奈瑟菌感染患者的脑脊液标本，TaqMan荧光定量PCR检测11份阳性，乳胶凝集方法检测6份阳性。结论TaqMan荧光定量PCR方法能特异地检测和鉴定不同血清群脑膜炎奈瑟菌，具有较高的灵敏性和快速检测的特点，能提高临床疑似脑膜炎奈瑟菌感染病例的阳性检出率。

Objective To establish TaqMan Real-Time PCR method for detection and identification of Neisseria meningitidis. Methods Seven sets of primers and FAM-labeled probes targeting different genes of Neisseria meningitidis were designed and synthesized, ctrA gene was used for identification of N. meningitidis species. Six serogruops （ A, B, C, X, Y, W^3s ） of N. meningitidis were detected with following genes： sacB （A）, siaD （B）, siaD （C）, xcbB （X）, synF （Y） and synG （W135） respectively. Sensitivity and specificity of Real-Time PCR were assessed for different primers and probes. 121 cerebrospinal fluid（CSF） specimens from suspected N. meningitidis invasive meningitis cases were detected by latex agglutination test and Real-Time PCR assay simultaneously. Results 79 N. meningitidis isolates of different serogroups could be detected and identified by seven sets of primers and probes in this study. Real-Time PCR seemed more sensitive than standard PCR by 101-103 times. The respective sensitivities for ctrA , sacB , siaD （ B ） , siaD （ C ） , xcbB , synF and synG were 8,8,80,8,8,80,8 genome DNA copies in each reaction. Of the 121 CSF specimens, 11 were positive for Real-Time PCR and 6 for latex agglutination test. Conclusion Real-Time PCR could rapidly detect and identify N. meningiti＇dis of different serogroups and seemed more sensitive. It could be widely used for diagnose of invasive meningitis caused by N. meningitidis.