结核杆菌耐链霉素Rrs基因点突变单侧延长臂发夹探针设计与检测研究

The study on detecting mutation site in Rrs gene of streptomycin resistant MTB by fair clamp probes with single extending arm technology

目的选择结核杆菌耐链霉素(Sm)Rrs基因包含主要突变位点的序列设计发夹探针及其单侧延长臂发夹探针,建立扩增体系及发夹探针芯片检测方法。方法运用Beacon designer软件设计Rrs基因包含主要突变位点的发夹探针及其单侧延长臂发夹探针,建立其扩增体系及发夹探针芯片检测方法,应用荧光显微镜检测荧光信号。结果通过荧光显微镜检测到标准株及耐SM株PCR产物与发夹探针杂交后荧光信号区别明显;51株耐链霉素(SM)与10株H37RV标准株对照组荧光信号强度比较,耐SM组Rrs基因突变检出率为29%(P<0.05、P<0.01)。结论发夹探针技术是一种具有高灵敏核酸点突变检测技术。单侧延长臂发夹探针对解决发夹探针在芯片表面固定难题提供了一种有效方法,采用荧光显微镜观测荧光信号具有更强的荧光信号识别、放大功能及结果判断更准确等优点。

Objective To design fair clamp probe and the probes with single extending arm of Rrs gene with main mutation site of streptomycin （SM） resistant mycobacterium tuberculosis （MTB） and its amplification system, meanwhile, to find the detection way by fair clamp probes chip. Methods The software, Beacon designer, was used to design the fair clamp probe and the probes with single extending arm of Rrs gene with main mutation site and the amplification system, and the fluorescence microscope and image analysis software were used to detect the fluorescent signals and make a qualitative judgment. Results The difference between PCR products from standard strain and SM resistant one was obvious in detecting the fluorescent signals by use of fluorescence microscope. The detection rate of resistant SM was about 29% （P〈0.05 and P〈0.01）. The fluorescent signal from the 51 SM resistant strains was much stronger than that in 10 H37RV standard strains. Conclusion The fair clamp probe technology is a sensitive assay in detecting nucleic acid point mutation. It is an effective way to solve the problem of modifying the fair clamp probe on the surface of chip by the probes with single extending arm. It has many advantages such as stronger fluorescent signal recognition and amplification and is more accurate in signal detection.