嗜水气单胞菌实时荧光三重TaqMan PCR快速检测体系的建立

Novel triplex real-time TaqMan PCR assay for the detection of Aeromonas hydrophila

目的建立针对嗜水气单胞菌的高灵敏、高特异的实时荧光三重TaqMan聚合酶链式反应(PCR)快速检测体系。方法根据嗜水气单胞菌的16SrDNA、气溶素基因(aerA)和丝氨酸蛋白酶基因(ahp)的特异性序列设计引物及TaqMan探针,利用高通量实时荧光PCR检测平台探讨该检测体系的灵敏度;用29种其他肠道致病菌及院内感染中常见的致病菌评价该检测体系的特异性。结果实时荧光三重TaqMan PCR快速检测体系对嗜水气单胞菌重组质粒的检测灵敏度为1×102拷贝/反应体系;对嗜水气单胞菌基因组的检测灵敏度为5×10-2pg/反应体系;该体系的特异性引物探针在检测29种其他肠道致病菌及院内感染中常见的致病菌时未出现假阳性,整个反应在2h内完成。结论本研究建立的实时荧光三重TaqManPCR检测体系可作为嗜水气单胞菌灵敏、特异、快速的检测方法,并同时评价嗜水气单胞菌的致病潜力。

To develop a sensitive and specific triplex real-time TaqMan polymerase chain reaction（PCR） assay for the detection of Aeromonas hydrophila,3 sets of primers and probes were designed based on the sequences of the 16S rDNA,aerolysin（aerA） and serine protease（ahp） gene.High throughput real-time PCR system was used to evaluate the sensitivity of the assay,and the specificity was evaluated by 29 other common enteropathogenic bacteria and some isolates causing nosocomial infection.The sensitivity of the triplex real-time PCR assay for testing of recombinant plasmids was 1×102 copies per reaction,and that for testing of the Aeromonas hydrophila genome DNA was 5×10-2 pg per reaction.No specific amplification was presented when the 29 other common enter pathogenic bacteria and some isolates causing nosocomial infection were tested.Furthermore,the assay could be finished within 2 hours.The triplex real-time TaqMan PCR assay developed in our study was sensitive,specific and rapid,which could be used to detect Aeromonas hydrophila in samples and evaluate the potentially pathogenicity of the isolates simultaneously.