量子点高效偶联DNA用于铜绿假单胞菌检测

Optimization of DNA probe and quantum dot concentration on Pseudomonas aeruginosa

目的建立量子点高效标记铜绿假单胞菌16srDNA的方法,为实现铜绿假单胞菌的快速、准确检测奠定基础。方法采用巯基法固定铜绿假单胞菌的16SrDNA P1探针于石英晶体金膜上,加入检测样本进行首次杂交,清洗后加入量子点标记的P2探针进行第2次杂交;最终通过荧光强度判断铜绿假单胞菌的浓度。结果不同的DNA∶QD比例可以产生不同的QD-DNA复合体,该复合体的荧光强度对最终检测结果具有极大影响;随着DNA∶QD的比例增加,其偶联效率也随之增加,当DNA∶QD的比例为100∶1时,其偶联基本实现饱和,偶联效率不再升高。结论该实验建立了一种新型的基于量子点检测铜绿假单胞菌16SrDNA的方法,为临床微生物的快速检测提供了一种新的技术方案。

OBJECTIVE To optimize the condition that DNA probe conjugates with quantum dots in marking 16s rDNA of Pseudomonas aeruginosa and to establish a novel quantum dot-based detection technique for rapid detection of P.aeruginosa.METHODS Hydrosulfide group was adopted to immobilize the 16S rDNA probes of P.aeruginosa on the gold membrane of quartz crystal.We added the target molecular to perform the first hybridization.Then quantum dot-labeled secondary probe was introduced into the detection well after carefully rinse.The total fluorescence intensity was scanned by fluorescence microscopy after the secondary rinse.RESULTS Different DNA∶QD ratio could provide different DNA conjugation efficiency,which played an important role in the later fluorescence intensity detection.We found the increased DNA∶QD ratio could significantly enhance the detectable fluorescence signals;when the ratio reached 100∶1,the fluorescence intensity did not increase any longer,indicating that this concentration was the optimized conjugation concentration.CONCLUSION We have successfully optimized the DNA∶QD ratio and constructed a quantum dot-based detection technique that can effectively detect P.aeruginosa,providing a promising method for rapid detection of microorganism in clinics.