School of Medicine, Nankai University, Tianjin 300071, China.
College of Life Sciences, Qingdao University, Qingdao 266071, China.
J Microbiol Methods. 2020 Apr;171:105866. doi: 10.1016/j.mimet.2020.105866. Epub 2020 Feb 11.
Vibrio parahaemolyticus, a major food-borne pathogen, is a gram-negative rod-shaped halophilic bacterium which inhabits marine environments throughout the world. It can pose a threat to humans after the consumption of raw or undercooked seafood. Fast detection is crucial for hindering and controlling V. parahaemolyticus infection. Compared with traditional methods, loop-mediated isothermal amplification (LAMP) is a simple, rapid and versatile method. It can be performed at one temperature without the need for cycling. As a new method in recent years, LAMP combined with a chromatographic flow dipstick (LFD) meets the needs of point-of-care testing without the need for special instruments. It avoids the limitations of LAMP, reduces detection time and increases detection accuracy. Our previous studies have suggested that the optimized LFD method can improve the sensitivity of LAMP detection and shorten the isothermal amplification time during the detection process. In the present study, two LAMP assays were improved to LFD methods, and a LFD targeting 16S23S rRNA gene internal transcribed spacer (ITS) of V. parahaemolyticus was developed. The lower limit for tlh, toxR, ITS LFD assays were detected as 3.1 × 10, 3.1 × 10, and 3.1 × 10 CFU respectively, whether in pure cultures or artificially contaminated food samples. The shortest amplification times at the limit of each assay were determined as 20 min, 35 min and 25 min. A heating block was used to perform two (tlh and ITS) LFD assays to detect 20 food samples. Compared to a standard method (GB 4789.7-2013 National Food Safety Standards, Food Microbiology Inspection, Vibrio parahaemolyticus test), tlh and ITS LFD assays showed more MPN (most probable number) results than that of culture. It demonstrated that the improved LFD technology can provide a simple and rapid detection method with high sensitivity and specificity for detection of V. parahaemolyticus.
副溶血性弧菌，一种主要的食源性病原体，是一种革兰氏阴性杆状嗜盐菌，栖息在世界各地的海洋环境中。它可以在食用生的或未煮熟的海鲜后对人类造成威胁。快速检测对于阻止和控制副溶血性弧菌感染至关重要。与传统方法相比，环介导等温扩增 (LAMP) 是一种简单、快速和通用的方法。它可以在一个温度下进行，无需循环。作为近年来的一种新方法，LAMP 与色谱流动纸条 (LFD) 结合满足了即时检测的需求，而无需特殊仪器。它避免了 LAMP 的局限性，缩短了检测时间，提高了检测准确性。我们之前的研究表明，优化的 LFD 方法可以提高 LAMP 检测的灵敏度，并在检测过程中缩短等温扩增时间。在本研究中，我们对两种 LAMP 检测方法进行了改进，开发了一种针对副溶血性弧菌 16S23S rRNA 基因内转录间隔区 (ITS) 的 LFD 检测方法。tlh、toxR 和 ITS LFD 检测的下限分别为 3.1×10、3.1×10 和 3.1×10 CFU，无论是在纯培养物还是人工污染的食物样本中。每种检测方法的最短扩增时间分别确定为 20 分钟、35 分钟和 25 分钟。使用加热块对两种（tlh 和 ITS）LFD 检测方法进行了 20 个食品样本的检测。与标准方法（GB 4789.7-2013 食品安全国家标准，食品微生物检验，副溶血性弧菌检验）相比，tlh 和 ITS LFD 检测方法的 MPN（最可能数）结果多于培养物。这表明改进的 LFD 技术可以为副溶血性弧菌的检测提供一种简单、快速、高灵敏度和特异性的检测方法。
