利用F_0F_1-ATPase分子马达技术检测志贺氏菌

The study of a detecting technology for Shigellabased on F_0F_1-ATPase molecular motor biosensor

利用载色体chromatophore上的F0F1-ATPase分子马达生物传感器,建立应用在食品检测中快速检测方法。首先从嗜热菌中提取载色体,合成生物素化宋内氏志贺氏菌IpaH探针,在载色体ATP合酶的ε亚基上连接ε亚基抗体-生物素-链霉亲和素-生物素-IpaH探针,将待测宋内氏志贺氏菌标准菌株和阴性对照分别与此生物传感器结合,比较其催化ATP合成10 min后的ATP产生量,进而对宋内氏志贺氏菌DNA进行检测。ATP合成的量可以通过luciferase-luciferin检测试剂所体现的荧光强度大小标定。结果表明,chroIpaH(连接在载色体chro上的IpaH探针)的浓度在0.031 mg/mL,宋内氏志贺氏菌DNA浓度在50 ng/mL为最适检测条件。通过与实际检测样品的传统检测方法及PCR检测方法对照,具有良好的检测符合性。

The core technology is the use of chromatophore on the F0F1-ATPase molecular motor biosensor for rapid detection of Shigellasonnei in the food. Specific IpaHprobe were connected with F0F1-ATPase＇ s ~ subunit using avidin-biotin system, and then biosensors were constructed, the test samples and negative sample, respectively, were combined with biosensors, to compare their catalytic ATP synthesis after 10 min. Shigellasonnei DNA in the samples could be tested. The amount of ATP synthesis could be detected by measuring the amount of H＋ , while the amount of H＋could be indicated by fluorescence intensity through luciferase-luciferin assay. The results of our experiments showed that optimum conditions for detection were the concentration of chrolpaH was 0.031 mg/mL and the concen- tration of Shigellasonnei DNA was 40 ng/mL. Results from the detection of actual samples were in good agreement with those from traditional detection methods and PCR detection method.