分子信标探针荧光芯片检测结核分支杆菌的实验研究

Detection of Mycobacterium tuberculosis by Fluorescence Chip of Molecular Beacon Probe

目的研究运用分子信标探针荧光芯片检测结核杆菌的一种新方法并优化杂交条件。方法设计出针对结核杆菌检测的特异性分子信标探针并运用于荧光芯片,提高杂交效率部分优化包括分子信标探针浓度及纯度;杂交温度、杂交时间、杂交液配方;4种分子对分子信标荧光信号比较。结果Mg2+终浓度为5 mmol/L时,PCR扩增效率最高,分子信标荧光强度最强;单侧延长臂分子信标采用淬灭分子中间标记,能很好的结合在芯片上;3种杂交液的杂交结果比较:0.15%SDS效果好于0.2%SDS,10×SSC效果好于5×SSC;Cy3-BDH分子信标探针浓度约15μmol/L、FAM-DABCLYE探针浓度约9μmol/L时,杂交反应达到7 h可检测到较强信号;Cy3-BDH组成分子信标荧光-淬灭效率较Cy3-DABCLYE高。结论单侧延长臂分子信标探针设计较巧妙地把分子信标高特异性、高敏感性的优势自然运用到芯片技术上,结核杆菌分子信标荧光芯片检测具有良好的检出率、特异性与敏感性。

OBJECTIVE To have a research on the detection of Mycobacteriurn tuberculosis by fluorescence chip of molecular beacon probe. METHODS Fluorescence chip for the detection of M. tuberculosis was designed, based on the better bonding force and specificity of molecular beacon and target molecule. Hybridization efficiency was optimized, which included the concentration 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 5 mmol/L and the amplification efficiency of PCR approached the highest. Half extended molecular beacon probe could bind nice with chip when it was middle labeled with quench group. The results of 3 hybridization solutions were as follows： 0. 15% SDS better than 0. 2% SDS , 10 X SSC better than 5 X SSC. Strong signal of hybridization reaction could be detected when the concentration of Cy3-BDH molecular beacon probe was 15 μmol/L, the concentration of FAM-DABCLYE probe was 9μmol/L and the reaction time was 7 h. The efficiency of fluorescence quenching was higher when the molecule was consisted of Cy3-BDH, other than Cy3-DABCLYE. CONCLUSIONS The design of half extended molecular beacon probe tactfully makes 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 M. tuberculosis has satisfactory detecting rate, specificity and sensibility.