Real-Time Recombinase Polymerase Amplification Assay for the Detection of Vibrio cholerae in Seafood
Real-Time Recombinase Polymerase Amplification Assay for the Detection of Vibrio cholerae in Seafood
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实时重组酶聚合酶扩增测定法检测海鲜中的霍乱弧菌
DOI：
10.1007/s12161-017-0820-7
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发表时间：
2017-08-01
影响因子：
2.9
通讯作者：
Zhang，Weijia
中科院分区：
文献类型：
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作者：
Tang，Yuyi;Cao，Yunqing;Zhang，Weijia
霍乱弧菌（Vibrio cholesterol）是危害性最大的弧菌，对水产养殖动物乃至人类都构成了严重的威胁。近年来，一些针对霍乱弧菌的遗传标记被报道，但其应用受到技术或自身的限制。外膜脂蛋白基因lolB已被确定为一个特异性标记，通过使用PCR和定量PCR方法。在这项研究中，我们建立了一个实时重组酶聚合酶扩增（RPA）检测靶向lolB基因，试图提供一个敏感，快速，可靠的检测方法霍乱弧菌。通过扩增标准质粒稀释液来确定RPA测定的灵敏度。在10 min内达到低至5个拷贝的检测限，低于qPCR的检测限（1.5 h内25个循环后10个拷贝）。RPA测定的重现性通过8个独立实验进行验证，标准回归线的R2值较高（0.97）。此外，用这两种方法从其他细菌、虾、蛤和鱼中提取的DNA证实了特异性。最后，使用实时RPA和qPCR定量虾样品中的霍乱弧菌，结果一致，而使用这两种方法从蛤和鱼样品中均未获得扩增。通过对天然海产品的定量检测，证实了该方法在食品工业中的适用性。
Vibrio cholerae, the most hazardous Vibrio species, is threatening aquacultured animals and even human beings. In recent years, a few genetic markers that target V. cholerae have been reported, but their application was limited by techniques or themselves. The outer membrane lipoprotein gene lolB has been identified as a specific marker by using PCR and quantitative PCR methods. In this study, we developed a real-time recombinase polymerase amplification (RPA) assay targeting lolB gene in an attempt to provide a sensitive, rapid, and reliable detection method of V. cholerae. The sensitivity of RPA assay was determined by amplifying the standard plasmid dilutions. The detection limit down to five copies was achieved within 10 min, which was lower than that of qPCR (ten copies after 25 cycles within 1.5 h). The reproducibility of RPA assay was verified by eight independent experiments, presenting a high R2 value of the standard regression line (0.97). In addition, the specificity was confirmed with DNA extracted from other bacteria, shrimps, clams, and fishes using both methods. Finally, V. cholerae in shrimp samples were quantified using real-time RPA and qPCR with consistent outcomes, while no amplification was obtained from clam and fish samples using both methods. The applicability in the food industry was confirmed by quantitative detection of natural seafood.