结核分枝杆菌Emb330codon分子信标杂交及荧光显微观测  

Detecting Micro-luminescence of 330 Codon in emb gene of Mycobacterium tuberculosis by Molecular Beacon Hybridization

作　　者：陈庆海[1] 刘星[2] 黄君富[1] 罗阳[1] 匡红[3] 府伟灵[1] 

机构地区：[1]第三军医大学西南医院检验科,重庆400038 [2]第三军医大学学员旅十队,重庆400038 [3]解放军第452医院医教部,四川成都610021

出　　处：《中华医院感染学杂志》2009年第14期1908-1910,共3页Chinese Journal of Nosocomiology

基　　金：国家863计划重大专项(2002AARZ2023;2007AA022005);国家自然科学基金(30571775);军队十一五课题(06G073);重庆市计生委课题(2008-5)

摘　　要：目的设计结核分枝杆菌耐乙胺丁醇embB330codon位点分子信标,运用荧光显微镜观测分子DNA探针与embB330codon扩增产物杂交后的荧光信号,比较该位点突变株与标准株。方法运用软件Beacondesigner设计embB基因包含330codon的分子信标,应用荧光显微镜检测embB330codon扩增片段与探针杂交后荧光信号,比较扩增产物测序结果。结果通过荧光显微镜观测到结核标准株及embB330codon突变株PCR产物与探针杂交后荧光信号差异有统计学意义;33株耐乙胺丁醇组与10株H37RV标准株对照组荧光信号强度比较,耐乙胺丁醇组embB330codon突变检出率为3%,测序法突变检出率为3%。结论分子信标技术可以有效检测embB330codon单碱基靶点突变;应用荧光显微镜观测荧光杂交信号非常灵敏。OBJECTIVE To design molecular beacon detecting embB330 codon of ethambutol-resistant Mycobacteriurn tuberculosis （MTB） ,meanwhile, and try to detect fluorescence of mutation site of embB330 codon in liquid by fluorescence microscope by compareing the mutation strains and standard strains. METHODS The software, Beacon designer,was used to design molecular beacon detecting embB330codon and detecting fluorescence signal from hybridization between the amplified product and probe by fluorescence microscope, and to confer to the sequencing results. RESULTS The difference between PCR products from standard strain and ethambutol-resistant one was obvious in detecting the fluorescent light by use of fluorescence microscope. We detected fluorescent light signal between the 33 ethambutol-resistant strains and 10 H37RV standard strains. The rate of ethambutolresistant strains was about 3% , and the rate of sequencing was about 3%. CONCLUSIONS The technology of molecular beacon effectually can detect mutation single base site of embB330codon. Fluorescence microscope owns characteristics such as high sensitiveness to detect the fluorescent light.

关 键 词：耐乙胺丁醇embB基因 330密码子点突变 分子信标 荧光显微镜 

分 类 号：R378.91[医药卫生—病原生物学]