Jiangsu Key Laboratory of Marine Biological Resources and Environment, Jiangsu Key Laboratory of Marine Pharmaceutical Compound Screening, Co-Innovation Center of Jiangsu Marine Bio-industry Technology, Jiangsu Ocean University, Lianyungang, China.
Key Laboratory of Zoonosis Research by Ministry of Education, College of Veterinary Medicine, Jilin University, Changchun, China.
J Food Sci. 2020 Jun;85(6):1834-1844. doi: 10.1111/1750-3841.15105. Epub 2020 May 25.
Vibrio parahaemolyticus is an important pathogenic bacterium in both food safety management and mariculture. Rapid and accurate detection technologies are critical for effective control of its outbreak and spreading. Conventional technologies and polymerase chain reaction (PCR)-based approaches have limited usage because of the requirement of laboratory instruments and trained personnel. Using the isothermal recombinase polymerase amplification (RPA) technology, several detection assays have been developed with added convenience. Combining the lateral flow strip (LFS) test with RPA can further simplify the detection. In this study, an improved RPA assay using LFS for visual detection of V. parahaemolyticus was developed. Primers were designed targeting the virulence genes and screened for amplification efficiency, nonspecific amplification, and primer-dimer formation. Probes were designed for the best primer pairs, and the weakness of LFS tests, being easily affected by primer-dependent artifacts, was overcome by sequence modifications on primers and probe. The RPA-LFS assay took 25 min at 35 to 45 °C, and showed excellent specificity. It detected as low as one colony forming unit (CFU) of V. parahaemolyticus per reaction without DNA purification, or 10 CFU/10 g spiked food samples with 2 hr of enrichment. The detection limit was better than the currently available RPA-based detection methods. Application of the RPA-LFS assay for simulated samples or real clinical samples showed accurate and consistent detection results compared to bioassay and quantitative PCR. The RPA-LFS assay provided a rapid, accurate, and convenient V. parahaemolyticus detection method suitable for on-site detection in resource-limited conditions. PRACTICAL APPLICATION: This research developed a rapid and visual detection technology for Vibrio parahaemolyticus that is not dependent on complicated equipment. The detection process takes 25 min and the result is read with the naked eye. A detection kit can be developed based on this technology for on-site detection of V. parahaemolyticus in resource-limited regions for food safety management and mariculture.
副溶血性弧菌既是食品安全管理又是海水养殖中的重要致病菌。快速准确的检测技术对于有效控制其爆发和传播至关重要。由于需要实验室仪器和经过培训的人员，常规技术和聚合酶链反应（PCR）为基础的方法的使用受到限制。使用等温重组酶聚合酶扩增（RPA）技术，已经开发了几种具有附加便利性的检测方法。将横向流动条（LFS）测试与 RPA 结合使用可以进一步简化检测。在这项研究中，开发了一种使用 LFS 进行副溶血性弧菌可视化检测的改进 RPA 检测方法。设计了针对毒力基因的引物，并筛选了扩增效率、非特异性扩增和引物二聚体形成。针对最佳引物对设计了探针，通过对引物和探针进行序列修饰克服了 LFS 测试容易受到引物依赖性伪影影响的弱点。RPA-LFS 检测在 35 至 45°C 下需要 25 分钟，显示出极好的特异性。它无需 DNA 纯化，即可检测到每反应低至一个副溶血性弧菌菌落形成单位（CFU），或在 2 小时富集后，检测到 10 克添加食品样品中的 10 CFU。检测限优于现有的基于 RPA 的检测方法。与生物测定和定量 PCR 相比，该 RPA-LFS 检测方法对模拟样品或实际临床样品的应用显示出准确且一致的检测结果。该 RPA-LFS 检测方法提供了一种快速、准确、方便的副溶血性弧菌检测方法，适用于资源有限条件下的现场检测。实际应用：本研究开发了一种不依赖复杂设备的副溶血性弧菌快速可视化检测技术。检测过程需要 25 分钟，结果用肉眼读取。可以基于该技术开发检测试剂盒，用于资源有限地区的食品安全管理和海水养殖中的副溶血性弧菌现场检测。
