摘要
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.
出处
《中华流行病学杂志》
CAS
CSCD
北大核心
2008年第4期360-364,共5页
Chinese Journal of Epidemiology
基金
国家“十五”科技攻关计划资助项目(2005BA711A09)