Ningbo University, Ningbo, 315211, China.
Marine Environmental Monitoring Center of Ningbo, State Oceanic Administration, China.
Mol Cell Probes. 2019 Apr;44:8-13. doi: 10.1016/j.mcp.2019.01.001. Epub 2019 Jan 3.
Vibrio harveyi is a pathogen that infects fish and shellfish worldwide, causing severe economic losses for the aquaculture industry. As the early diagnosis of V. harveyi infection is crucial to disease surveillance and prevention in cultured marine animals, a fast and accurate method to detect V. harveyi is required. Here, we performed recombinase polymerase amplification (RPA) using novel primers specifically designed to recognize the V. harveyi toxR gene, which encodes a transmembrane protein, and then hybridized this gene with a carboxy fluorescein (FAM)-labeled probe. The optimal conditions for the real-time RPA assay were a probe concentration of 90 nM and a 20 min incubation at 37 °C. The sensitivity of our real-time RPA assay was 50 copies of the standard plasmid, while that of real-time PCR was 500 copies. In V. harveyi-spiked Pseudosciaena crocea samples, the sensitivity of our real-time RPA was 60 CFUs per reaction, while that of PCR was 600 CFUs per reaction. SPSS probit regression analysis indicated that the limit of detection (LOD) of our RPA assay, with 95% probability, was 18 copies. The LOD was reached within 20 min and was highly reproducible across eight independent assays. Our novel RPA method successfully differentiated V. harveyi from all other tested Vibrio species, including some that were closely related. Our real-time RPA assay, in combination with a rapid DNA extraction protocol, is a fast and accurate tool for the detection of V. harveyi and for monitoring disease outbreaks. This tool will be valuable for the aquaculture industry.
哈维弧菌是一种感染鱼类和贝类的病原体，在世界范围内造成水产养殖业的严重经济损失。由于早期诊断哈维弧菌感染对于养殖海洋动物的疾病监测和预防至关重要，因此需要一种快速准确的方法来检测哈维弧菌。在这里，我们使用专门设计的识别哈维弧菌 toxR 基因的新型引物进行重组酶聚合酶扩增（RPA），该基因编码一种跨膜蛋白，然后将该基因与羧基荧光素（FAM）标记的探针杂交。实时 RPA 检测的最佳条件是探针浓度为 90 nM，37°C 孵育 20 分钟。我们的实时 RPA 检测的灵敏度为 50 个标准质粒拷贝，而实时 PCR 的灵敏度为 500 个拷贝。在哈维弧菌感染的青石斑鱼样品中，我们的实时 RPA 的灵敏度为每个反应 60 CFU，而 PCR 的灵敏度为每个反应 600 CFU。SPSS 概率单位回归分析表明，我们的 RPA 检测的检测限（LOD）为 95%概率时为 18 个拷贝。LOD 在 20 分钟内达到，并且在八个独立的检测中具有高度重现性。我们的新型 RPA 方法成功地区分了哈维弧菌和所有其他测试的弧菌属物种，包括一些密切相关的物种。我们的实时 RPA 检测与快速 DNA 提取方案相结合，是一种快速准确的检测哈维弧菌和监测疾病爆发的工具。该工具将对水产养殖业具有重要价值。
