环介导等温扩增技术在嗜水气单胞菌检测中的应用

Loop-mediated isothermal amplification assay for the detection of Aeromonas hydrophila

目的建立环介导等温扩增技术(Loop-mediated isothermal amplification,LAMP)对嗜水气单胞菌检测的方法,并用临床标本对该方法进行评价。方法根据嗜水气单胞菌desA基因的保守序列设计特异性引物,利用参考质粒评价该检测体系的灵敏度;用30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;用粪便模拟样本以及临床样本评价该检测体系的临床检测的可行性。结果 LAMP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;LAMP检测体系在检测30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;LAMP检测体系在粪便模拟标本中的检测下限为5×103 CFU/g;通过与qPCR技术相比较,LAMP具有更高的灵敏度以及更优秀的临床标本的检测能力。结论本研究建立的LAMP方法是首次将LAMP技术运用到了嗜水气单胞菌的检测,所需仪器设备简单,可作为一种快速的筛查方法在临床检验和基层实验室应用。

To develop a sensitive and specific loop mediated isothermal amplification（LAMP） assay for the detection of Aer- omonas hydrophila, a set of 6 primers targeted toward the desA gene of the A. hydrophila was designed using PrimerExplorer V4 software（Eiken Chemical Co. Ltd. , Tokyo, Japan） based on the conserved sequences. The performance of the assay with reference plasmids, simulated human stools, and clinical specimens were evaluated and compared with quantitative PCR （qPCR）. No false positive results were observed for the 33 non-A, hydrophila strains used to evaluate assay specificity. The sensitivity of the LAMP assay was approximately 20 copies per reaction in reference plasmids and 5 × 10^3 CFU per gram in spiked human stool, which were more sensitive than the results of qPCR. The performance of the LAMP assay was evaluated with clinical specimens, which had the better detection ability than qPCR. In conclusion, the LAMP assay developed in this study is a valuable method for rapid, cost-effective, and simple detection of A. hydrophila in basic clinical and field laboratories.