MGB探针实时定量PCR检测致病性嗜水气单胞菌

摘要

[Objective] A real-time PCR, based on TaqMan hybridization probes technology, were developed and applied to detect pathogenic Aeromonas hydrophila.One pairs of primer and one probes were designed for detection aerolysin genes.The aerolysin probe was 5′end labeled with FAM and 3′end labeled with TaqMan-MGB.The detection limits of the sensitivity assays were 1.25×100 CFU/μl;the qualitative consensus PCR assay indicated all Aeromonas hydrophila were found positive and did not detect DNA from non-Aeromonas hydrophila isolates.In the duplicated experiment, coefficients of variation intra-assay and inter-assay over the dynamic range of the MGB probe assays were lower than 3% and 5%, respectively.These results showed that this real-time PCR can detect the Aeromonas hydrophila from samples rapidly.In conclusion, this real-time PCR-based method is rapid, sensitive and specific for the detection of pathogenic Aeromonas hydrophila , and it is a practical method for pathogenic Aeromonas hydrophila detection.%以嗜水气单胞菌气溶素(aerolysin)基因为待检靶基因,设计一对引物和一条MGB(Minor Groove Binder)探针,Aero 基因探针5′端用FAM基团标记,3′端用TaqMan-MGB标记.建立并优化了检测嗜水气单胞菌荧光定量PCR方法,可检测的最低细菌数为1.25×100 CFU/μl;试验中嗜水气单胞菌检测结果均为阳性,而非嗜水气单胞菌检测结果均为阴性;重复性试验中,批间差异小于5%,批内差异小于3%.试验结果显示,荧光定量PCR方法可对致病性嗜水气单胞菌株进行快速鉴定.该方法的建立为致病性嗜水气单胞菌的检测提供了一种简便、快速的途径,是一种比较实用的致病性嗜水气单胞菌的检测方法.