| 娄静,刁保卫,王洪霞,崔志刚,祁国明,阚飙.扩增片段长度多态性分析用于霍乱弧菌分子分型的方法建立和应用评价[J].Chinese journal of Epidemiology,2007,28(6):580-585 |
| 扩增片段长度多态性分析用于霍乱弧菌分子分型的方法建立和应用评价 |
| Establishment of fluorescent amplified fragment length polymorphism in VIbroi cholera and evaluation in molecular typing |
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| DOI： |
| KeyWord: 霍乱弧菌 扩增片段长度多态性 脉冲场凝胶电泳 分子分型 |
| English Key Word: Ⅵbrio chooloera Amplified fragment length polymorphism Pulsed-field gelelectrop}10resis IⅥolecular tvping |
| FundProject:国家“十五”科技攻关计划课题资助项目(2003BA712A05一04) |
| Author Name | Affiliation | E-mail | | LO Jing | Sdate Key Loaboratory for infectious Disease and Control and Prevention, Chinese Centre for Disease Control and Prevention, Beijing 102206, China | | | DIAO Bao-jing | Sdate Key Loaboratory for infectious Disease and Control and Prevention, Chinese Centre for Disease Control and Prevention, Beijing 102206, China | | | WANG Hong-xia | Sdate Key Loaboratory for infectious Disease and Control and Prevention, Chinese Centre for Disease Control and Prevention, Beijing 102206, China | | | CUI Zhi-gang | Sdate Key Loaboratory for infectious Disease and Control and Prevention, Chinese Centre for Disease Control and Prevention, Beijing 102206, China | | | QI Guo-ming | Sdate Key Loaboratory for infectious Disease and Control and Prevention, Chinese Centre for Disease Control and Prevention, Beijing 102206, China | | | KAN Biao | Sdate Key Loaboratory for infectious Disease and Control and Prevention, Chinese Centre for Disease Control and Prevention, Beijing 102206, China | kanbiao@icdc.cn |
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| Abstract: |
| 目的建立霍乱弧菌的基于红外荧光标记引物的扩增片段长度多态性(AFLP)分型方法, 评价脉冲场凝胶电泳(PFGE)和AFLP对霍乱弧菌的分型能力。方法选择PFGE带型均不相同的47株霍乱弧菌作为评价菌株, 建立AFI。P对于霍乱弧菌的分型方法;确定操作程序后选择83株分离自1962-2005年11个省的霍乱弧菌, 进行AFI。P和PFGE对于霍乱弧菌分型能力的比较。AFI。P分型方法采用了红外荧光标记PCR引物, 利用LI-COR4300自动测序仪完成电泳、Saga“软件对电泳图谱进行编辑。PFGE采用PulseNet提供的标准化程序。结果AFI, P用于霍乱弧菌分型时选择性引物携带碱基数为1, 最优引物组合为EcoR I-G/M”I-T, AFLP分型实验的重复性达到99.2%。AFI。P将83株霍乱弧菌分为52个型别, D值为0.9545;PFGE将此83株菌分为44种带型, D值为0.9251。结论优化固定了AFLP对于霍乱弧菌的分型程序, 重复性好;AFLP比PFGE具有更好的分型能力, 能够用于分离菌株的分子分型。结合已成为实验室网络监测标准分析方法的PFGE, 可对分离株做更细致的分型比较。 |
| English Abstract: |
| ObjectiveTo develop fluorescent amplified fragment length polynlorphism(AFI。P)method and to evaluate the its typing capability with pulsed-field gel electrophoresis(PFGE)in molecular typing of VIbrio chloerae.MethodsForty-seven strains of V.cholerae, with different PFGE patterns, were selected as the reference group to optimize the selective primers of AFLP analysis.Eighty-three strains including 20 strains from one epidemic episode, isolated from different provinces during 1961 and 2005, were used to compare the typing abilities of AFLP and PFGE.LI-COR4300 DNA sequencing system was used for AFLP electrophoresis.The images were recorded by SagaMX Software and transferred to BioNumerics for clustering analysis.A standard protocol for V.cholerae from Pulsenet was used in PFGE.ResultsWhen comparison was made with different selective primers on AFLP based on the 47 strains, results showed that the optimized selective primer pair was EcoR I-G/M5e I-T, and the repoducibility of the tests was 99.2%.Eightv-three isolates showed 5 2 AFLP patterns and 44 PFGE patterns, with D values as O.9545(AFLP)and O.9251(PFGE)respectively.ConclusionThe protocol of fluorescent AFLP on V.cholerae typing was established.AFLP waS higher than PFGE in discrimination of V.Cholerae which could be used for molecular typing. When combined with PFGE, AFLP became a more insightful tool to identify genome difference of different is01ates. |
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