PCR产物与结核分支杆菌分子信标杂交优化及荧光检测的初步实验研究

Investigation on the optimizing of the hybridization of PCR product with the molecular beacon of bacillus tuberculosis and the fluorescence detection of bacillus tuberculosis

目的建立PCR反应后再加入结核分支杆菌分子信标杂交试验,优化杂交条件。并对分子信标PCR杂交产物荧光信号的观测条件进行摸索。方法通过对杂交温度、杂交时间以及杂交方式的优化,建立PCR反应后再加入TB分子信标杂交试验。运用荧光分光光度计和荧光显微镜对MB PCR杂交产物荧光信号的观测条件进行摸索。并进行不同荧光-淬灭分子对标记分子信标的观测试验。结果PCR反应后再加入TB分子信标杂交试验中水浴、PCR仪、恒温摇床的最适杂交温度和适杂时间分别为:55℃,6h;55℃,4h;50℃,7h。阴-阳性效果区分;水浴>恒温摇床>PCR仪。荧光分光光度仪检测MB PCR杂交阳性产物、MB PCR杂交阴性产物(茎环结构未打开)、无游离分子信标探针PCR产物(空白对照)三者的荧光光亮度比值为:6.102/0.136/0.003。荧光显微镜观测MB PCR杂交阳性产物、MB PCR杂交阴性产物(茎环结构未打开)荧光强度明显区别。本试验Cy3-BDH组成分子信标荧光-淬灭效率较Cy3-DABCLYE高,FAM-DABCLYE组成分子信标荧光-淬灭效率较FAM-BDH高。结论建立起结核分支杆菌分子信标荧光芯片的检测技术,包括样本制备、芯片制备、杂交反应以及扫描和图像分析等方法。并兼顾到各种因素对单侧延长臂分子信标芯片的影响,对反应体系中各条件进行了合理的优化,使所建立的检测体系稳定可靠。

Objective To have a research on the detection of bacillus tuberculosis by fluorescence chip of molecular beacon probe. Methods Fluorescence chip for the detection of bacillus tuberculosis was designed, based on the better bonding force and specificity of molecular beacon and target molecule. Hybridization efficiency was optimized, which includes the concen-tration and purity of molecular beacon probe, hybridization temperature, hybridization time ,the prescription of hybridization solution and the comparing of molecular beacon fluorescence signals of 4 molecules. Results Molecular beacon fluorescence signal approached the highest when the final concentration of Mg was 5mmol/L and the amplification efficiency of PCR approached the highest, haft extended molecular beacon probe could bind nice with chip when it was middle labeled with quench group. The resuits of 3 hybridization solution were as follows： 0.15%SDS better than 0.2%SDS, 10×SSC better than 5×SSC. Strong signal of hybridization reaction could be detected when the concentration of Cy3-BDH molecular beacon probe was 15μmol/L, the con- centration of FAM-DABCLYE probe was 9μmol/L and the reaction time was 7h. The efficiency of fluorescence quenching was higher when the molecule was consisted of Cy3-BDH,other than Cy3-DABCLYE. Conclusion The design of half extended molecular beacon probe tactfully make use of the advantages of the high bonding force and specificity of molecular beacon probe on the chip technology. Fluorescence chip of molecular beacon for bacillus tuberculosis has satisfactory rate, specificity and sensibility.