EMB基因330密码子分子DNA探针设计及荧光检测

Fluorescence detection and molecular DNA probe design of 330 codon in embB gene

目的针对结核杆菌耐乙胺丁醇embB330密码子位点设计分子DNA探针,尝试运用荧光分光光度计直接观测液相中分子DNA探针与embB330密码子扩增产物杂交后的荧光信号,从而检出该位点突变。方法运用软件Beacon designer设计embB基因包含330密码子的分子DNA探针,应用荧光分光光度计检测embB306密码子扩增片段与探针杂交后荧光信号,比较扩增产物测序结果。结果通过荧光分光光度计观测到结核标准株及embB330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embB330密码子突变检出率为3%,测序法突变检出率为3%。结论分子DNA探针技术可以有效检测embB330密码子单碱基靶点突变;应用荧光分光光度计直接观测液相荧光杂交信号简单、灵敏。

Objective To design molecular DNA probe detecting embB330 codon of Ethambutol resistant mycobacterium tuberculosis （ MTB）, and to detect fluorescence of mutation site of embB330 codon in liquid by fluorescence spectrophotometer. Methods The software, beacon designer, was used to design molecular DNA probe detecting embB306 codon and detecting fluorescence signal from hybridization be- tween the amplified product and probe by fluorescence spectrophotometer, and confer to the sequencing results. Results The difference between PCR products from standard strain and Ethambutol resistant one is 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 resistant Ethambutol detected is about 3%, and the rate of sequencing is about 3%. Conclusion The technology of molecular DNA probe can effectually detect a mutation single basyl site of embB 330 codon. Fluorescence spectrophotometer have characteristics such as high sensitive- ness, simple to directly detect the fluorescent light in liquid.