发夹DNA探针检测乙胺丁醇耐药结核杆菌embB基因306密码子突变研究

The study on detecting mutation site of 306 codon in Ethambutol resistant embB gene of Mycobacterium tuberculosis bacterium by hair clamp probe

目的:针对结核杆菌耐乙胺丁醇embB306codon位点,设计发夹DNA探针及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,并初步运用荧光显微镜观测探针与扩增产物杂交的荧光信号。方法:运用Beacondesigner软件,设计embB基因包含306codon的发夹DNA探针及其相应的单侧延长臂发夹DNA探针,荧光显微镜检测embB306codon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embB306codon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embB306codon突变检出率为66%,测序法突变检出率为69%。结论:embB306codon点突变是结核杆菌耐乙胺丁醇的主要原因;发夹DNA探针芯片技术可以有效检测单碱基靶点突变;应用荧光显微镜可以高灵敏地观测荧光芯片杂交靶点。

Objective：To design hair clamp probe and the probes with single extending arm detecting embB306codon of Ethambutol resistant Mycobacterium tuberculosis bacterium （ MTB ）, meanwhile, to design fair clamp probe chip and detecting fluorescence signal from hybridization between the amplified product and probe by fluorescence microscope. Methods：The software, Beacon designer, was used to design fair clamp probe and the probes with single extending arm detecting embB303codon and detecting 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 resistant ethambutol detected with hair clamp probe was about 66%, and the rate of sequencing was about 69%. Conclusions：The mutation site of embB306codon of MTB is the main reason of ethambutol resistant MTB. The technology of fair clamp DNA probe chip can effectively detect mutation of single base site. Fluorescence microscope can sensitivily detect the site of hybridization on fluorescence chip.