耐甲氧西林金黄色葡萄球菌mecA基因分子探针杂交荧光观测

Detection of mecA gene with fluorescence of hybridization with molecular probe in methicillin-resistant staphylococcus aureus

目的针对耐甲氧西林金黄色葡萄球菌mecA基因扩增片段,设计一种茎环结构的DNA分子探针,运用荧光显微镜观测DNA分子探针与mecA基因扩增产物杂交后的荧光信号。方法采用Beacon designer 7.0软件对mecA基因的特异性扩增片段设计茎环结构的DNA分子探针,应用荧光显微镜检测mecA基因扩增片段与探针杂交荧光信号。结果通过荧光显微镜观测出28株耐甲氧西林金黄色葡萄球菌和8株铜绿假单胞菌对照菌的特异性扩增片段,与DNA分子探针杂交后,荧光信号强度差异有统计学意义;该杂交条件优化:50℃为最佳杂交温度;10 h为最佳杂交时间。结论茎环结构DNA分子探针技术,结合荧光显微镜可高灵敏检测mecA基因的扩增片段,mecA基因是金黄色葡萄球菌耐甲氧西林的主要原因。

OBJECTIVE To design stem-ring molecular probe detecting the amplified product of mecA gene of methicillin-resistant Staphylococcus aureus,meanwhile,to try to detect fluorescence of hybridization between the molecular probe and the amplified product of mecA gene by fluorescence microscope.METHODS The software,Beacon designer,was used to design stem-ring molecular probe detecting the amplified product of mecA gene and detecting fluorescence signal from hybridization between the amplified product and probe by fluorescence microscope.RESULTS The difference between the amplified products of 28 strains MRSA and 8 strains Pseudomonas aeruginosa was significant in detecting the fluorescent light by use of fluorescence microscope.The best hybridization temperature and hybridization time were 50℃ and 10 hours respectively.CONCLUSION The technology of tem-ring molecular probe combined with fluorescence microscope highly sensitive can detect the amplified product of mecA gene.The mecA gene in S.aureus is the main reason leads to methicillin-resistance.