量子点荧光双杂交技术检测铜绿假单胞菌的实验研究

Rapid detection of pseudomonas aeruginosa with quantum dot based dual-hybirdization technique

目的建立量子点荧光双杂交核酸检测平台,并实现铜绿假单胞菌(PAE)的快速、准确检测。方法采用巯基法固定铜绿假单胞菌的16S rDNA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第二探针进行第2次杂交,最终通过荧光强度以判断PAE是否存在。结果该量子点双杂交检测平台具有良好的性噪比,其荧光强度与扩增产物的稀释倍数成线性负相关,当PAE扩增产物的稀释倍数在<10 000倍时具有良好的线性曲线;特异性实验表明,该方法可以将PAE与同源的多种细菌相区分,证明了其高度特异性,方法学比较实验证明,双探针杂交方法的灵敏度和特异性均>90.0%,已能初步满足临床检测需要。结论该量子点荧光双杂交核酸检测平台,可有效将PAE与同源的多种细菌相区分,为临床微生物的快速检测提供了一种新的技术方案。

OBJECTIVE To establish a novel quantum dot-based dual-hybirdization detection technique and apply it for the rapid detection of Pseudomonas 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 were introduced into the detection well after carefully rinse.The total fluorescence intensity was scanned by fluorescence microscopy after the secondary rinse.RESULTS The quantum dot based microorganism biosensor had good signal-noise ratio.The fluorescence intensity was proportioned to the concentration diluted PCR products.A good relationship was evidenced between the fluorescence intensity and PCR products in the period of less than 10000 fold dilution.Specificity experiments had also showed that the P.aeruginosa could be easily differentiated from other homologous bacteria.Methodological experiments had also proved the high sensitivity and specificity（both higher than 90.0%）.CONCLUSION This newly constructed quantum dot-based dual-hybirdization detection technique can effectively detect P.aeruginosa,providing a promising method for rapid detection of microorganism.