| 朱兵清,徐丽,李马超,任红宇,田国忠,高源,王艳华,祁国明,阚飙,邵祝军.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[J].Chinese journal of Epidemiology,2008,29(4):360-364 |
| 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 application of TaqMan Real-Time PCR in detection and serogrouping of Neisseria meningitidis |
| Received:December 06, 2007 |
| DOI： |
| KeyWord: 脑膜炎奈瑟菌 TaqMan 荧光定量PCR |
| English Key Word: Neisseria meningitidis TaqMan Real-Time PCR |
| FundProject:国家"十五"科技攻关计划资助项目(2005BA711A09) |
| Author Name | Affiliation | E-mail | | ZHU Bingqing | State Key Laboratory for Infectious Disease Control and Prevention, National Institute for Communicable Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing 102206, China | | | XU Li | State Key Laboratory for Infectious Disease Control and Prevention, National Institute for Communicable Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing 102206, China | | | LI Machao | State Key Laboratory for Infectious Disease Control and Prevention, National Institute for Communicable Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing 102206, China | | | REN Hongyu | State Key Laboratory for Infectious Disease Control and Prevention, National Institute for Communicable Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing 102206, China | | | TIAN Guozhong | State Key Laboratory for Infectious Disease Control and Prevention, National Institute for Communicable Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing 102206, China | | | GAO Yuan | State Key Laboratory for Infectious Disease Control and Prevention, National Institute for Communicable Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing 102206, China | | | WANG Yanhua | State Key Laboratory for Infectious Disease Control and Prevention, National Institute for Communicable Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing 102206, China | | | QI Guoming | State Key Laboratory for Infectious Disease Control and Prevention, National Institute for Communicable Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing 102206, China | | | KAN Biao | State Key Laboratory for Infectious Disease Control and Prevention, National Institute for Communicable Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing 102206, China | | | SHAO Zhujun | State Key Laboratory for Infectious Disease Control and Prevention, National Institute for Communicable Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing 102206, China | shaozhujun@icdc.cn |
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| Abstract: |
| 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101～103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。 |
| English Abstract: |
| 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,W135)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. 121cerebrospinal fluid(CSF)specimens from suspected N.meningitidis invasive meningitis cases were detected by latex agglutination test and Real-Time PCR assay simultaneously.Resuits 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 bv 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 genomeDNA 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.meningitidis of different serogroups and seemed more sensitive.It could be widely used for diagnose of invasive meningitis caused bv N.meningitidis. |
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