Kanoktippornchai Boonnapa, Chomvarin Chariya, Engchanil Chulapan, Wongboot Warawan
Southeast Asian J Trop Med Public Health. 2014 Mar;45(2):375-82.
Abstract. Detection of toxigenic Vibrio cholerae O1/O139 in aquatic environment is difficult to achieve using the culture method. For direct detection of viable toxigenic V. cholerae in aquatic environment, we developed a triplex reverse transcription (RT)-PCR, targeting genes for the outer membrane protein (ompW), cholera toxin A (ctxA) and toxin-coregulated pilli (tcpA) and compared the assay with the culture method. After enrichment of the bacteria-containing filters in alkaline peptone water for 6 hours, the sensitivity of triplex RT-PCR for detecting V. cholerae was 7 cfu/ml. Of the 80 environmental water samples collected from various regions in Northeast Thailand, triplex RT-PCR detected 15 toxigenic and 20 non-toxigenic V. cholerae, whereas the culture method detected only 3 toxigenic V. cholerae--containing water samples. These results show that this triplex RT-PCR method could be used as an alternative tool for rapid and sensitive identification of viable toxigenic V cholerae in environmental water samples.
摘要。利用培养方法难以在水生环境中检测产毒霍乱弧菌O1/O139。为了直接检测水生环境中存活的产毒霍乱弧菌，我们开发了一种三重逆转录（RT）-PCR方法，该方法针对外膜蛋白（ompW）、霍乱毒素A（ctxA）和毒素协同调节菌毛（tcpA）的基因，并将该检测方法与培养方法进行了比较。在碱性蛋白胨水中将含细菌的滤膜富集6小时后，三重RT-PCR检测霍乱弧菌的灵敏度为7 cfu/ml。从泰国东北部不同地区采集的80份环境水样中，三重RT-PCR检测到15份产毒霍乱弧菌和20份非产毒霍乱弧菌，而培养方法仅检测到3份含有产毒霍乱弧菌的水样。这些结果表明，这种三重RT-PCR方法可作为一种替代工具，用于快速、灵敏地鉴定环境水样中存活的产毒霍乱弧菌。
