CAJ | 학술논문

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.
이기수기단포균기용소(aerolysin)기인위대검파기인,설계일대인물화일조MGB(Minor Groove Binder)탐침,Aero 기인탐침5′단용FAM기단표기,3′단용TaqMan-MGB표기.건립병우화료검측기수기단포균형광정량PCR방법,가검측적최저세균수위1.25×100 CFU/μl;시험중기수기단포균검측결과균위양성,이비기수기단포균검측결과균위음성;중복성시험중,비간차이소우5%,비내차이소우3%.시험결과현시,형광정량PCR방법가대치병성기수기단포균주진행쾌속감정.해방법적건립위치병성기수기단포균적검측제공료일충간편、쾌속적도경,시일충비교실용적치병성기수기단포균적검측방법.
[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.