乙胺丁醇耐药基因embB突变的杂交荧光显微观测

Hybridization Fluorescence Microscopy of Ethambutol-resistant embB Gene Mutation

目的针对耐乙胺丁醇(EMB)的结核分枝杆菌embB303密码子位点,设计扩增引物及特异性发夹DNA探针其相应的单侧延长臂发夹DNA探针,建立发夹DNA探针芯片技术,并运用荧光显微镜观测发夹DNA探针与扩增产物的杂交荧光信号。方法运用Beacon designer软件,设计embB基因包含303密码子的发夹DNA探针,荧光显微镜检测embB303密码子扩增片段与发夹DNA探针杂交后荧光信号,扩增产物测序结果作比较。结果通过荧光显微镜观测到结核标准株及embB303密码子突变株PCR产物与探针杂交后荧光信号存在显著差异;33株耐EMB组与10株H37RV标准株对照组荧光信号强度比较,耐EMB组embB303密码子突变检出率为7%,测序法突变检出率为6%。结论应用荧光显微镜可以高灵敏观测发夹DNA探针与靶序列的杂交荧光信号,从而有效检测单碱基突变;embB303密码子点突变不是结核分枝杆菌耐EMB的主要原因。

OBJECTIVE To design specific amplification primer and hairpin DNA probe and probe with single extending arm detecting embB 303 codon of ethambutol-resistant Mycobacterium tuberculosis （MTB）, meanwhile, to detect fluorescence signal from hybridization between the amplified product and hairpin DNA probe by fluorescence microscope. METHODS The software, Beacon designer, was used to design hairpin DNA probe detecting embB 303 codon and fluorescence signal from hybridization between the amplified product and probe,and 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 with fluorescence microscope. We detected fluorescent light signal between the 33 ethambutol-resistant strains and 10 H37RV standard strains. The rate of ethambutol resistantee＇detected with hairpin DNA probe was about 7 %, and the rate of sequencing was about 6 %. CONCLUSIONS Fluorescence microscope can sensitively detect fluorescence signal from hybridization between hairpin DNA probe and target DNA, and effectively detect mutation of single base site. The mutation site of embB 303 codon of MTB is not the main reason of ethambutol resistant MTB.