Tanaka Natsuko, Iwade Yoshito, Yamazaki Wataru, Gondaira Fumio, Vuddhakul Varaporn, Nakaguchi Yoshitsugu, Nishibuchi Mitsuaki
Graduate School of Medicine, Kyoto University, Yoshida, Sakyo-ku, Kyoto 606-8501, Japan.
Mie Prefecture Health and Environment Research Institute, Sakura-cho, Yokkaichi-shi, Mie 512-1211, Japan.
J Food Prot. 2014 Jul;77(7):1078-85. doi: 10.4315/0362-028X.JFP-13-536.
Although thermostable direct hemolysin-producing (tdh(+)) Vibrio parahaemolyticus is the leading cause of seafood-borne gastroenteritis, the enumeration of tdh(+) V. parahaemolyticus remains challenging due to its low densities in the environment. In this study, we developed a most-probable-number (MPN)-based procedure designated A-IS(1)-LAMP, in which an immunomagnetic separation (IMS) technique targeting as many as 69 established K antigens and a loop-mediated isothermal amplification (LAMP) assay targeting the thermostable direct hemolysin (tdh) gene were applied in an MPN format. Our IMS employed PickPen, an eight-channel intrasolution magnetic particle separation device, which enabled a straightforward microtiter plate-based IMS procedure (designated as PickPen-IMS). The ability of the procedure to quantify a wide range of tdh(+) V. parahaemolyticus levels was evaluated by testing shellfish samples in Japan and southern Thailand, where shellfish products are known to contain relatively low and high levels of total V. parahaemolyticus, respectively. The Japanese and Thai shellfish samples showed, respectively, relatively low (< 3 to 11 MPN/10 g) and considerably higher (930 to 110,000 MPN/10 g) levels of tdh(+) V. parahaemolyticus, raising concern about the safety of Thai shellfish products sold to domestic consumers at local morning markets. LAMP showed similar or higher performance than conventional PCR in the detection and quantification of a wide range of tdh(+) V. parahaemolyticus levels in shellfish products. Whereas a positive effect of PickPen-IMS was not observed in MPN determination, PickPen-IMS was able to concentrate tdh(+) V. parahaemolyticus 32-fold on average from the Japanese shellfish samples at an individual tube level, suggesting a possibility of using PickPen-IMS as an optional tool for specific shellfish samples. The A-IS(1)-LAMP procedure can be used by any health authority in the world to measure the tdh(+) V. parahaemolyticus levels in shellfish products.
尽管产耐热直接溶血素（tdh(+)）的副溶血性弧菌是食源性肠胃炎的主要病因，但由于其在环境中的密度较低，对tdh(+)副溶血性弧菌进行计数仍然具有挑战性。在本研究中，我们开发了一种基于最大可能数（MPN）的方法，称为A-IS(1)-LAMP，其中针对多达69种已确定的K抗原的免疫磁珠分离（IMS）技术和针对耐热直接溶血素（tdh）基因的环介导等温扩增（LAMP）检测以MPN形式应用。我们的IMS采用了PickPen，一种八通道溶液内磁珠分离装置，它实现了基于微量滴定板的直接IMS程序（称为PickPen-IMS）。通过检测日本和泰国南部的贝类样本评估了该方法对广泛tdh(+)副溶血性弧菌水平进行定量的能力，在日本和泰国南部，已知贝类产品中总副溶血性弧菌含量分别相对较低和较高。日本和泰国的贝类样本分别显示出相对较低（<3至11 MPN/10 g）和相当高（930至11,000 MPN/10 g）的tdh(+)副溶血性弧菌水平，这引发了对在当地早市销售给国内消费者的泰国贝类产品安全性的担忧。在贝类产品中tdh(+)副溶血性弧菌广泛水平的检测和定量方面，LAMP显示出与传统PCR相似或更高的性能。虽然在MPN测定中未观察到PickPen-IMS的积极作用，但PickPen-IMS能够在单个试管水平上平均将tdh(+)副溶血性弧菌从日本贝类样本中浓缩32倍，这表明PickPen-IMS有可能作为特定贝类样本的可选工具。世界上任何卫生当局都可以使用A-IS(1)-LAMP程序来测量贝类产品中tdh(+)副溶血性弧菌的水平。
