Department of Biology, Faculty of Science, Srinakharinwirot University, Bangkok, Thailand.
Lett Appl Microbiol. 2010 Jan;50(1):36-42. doi: 10.1111/j.1472-765X.2009.02749.x.
The present study was aimed to develop a loop-mediated isothermal amplification (LAMP) assay for rapid and specific detection of Vibrio cholerae.
A set of five designed primers that recognized specifically the V. cholerae ompW gene was used. The optimized time and temperature conditions for the LAMP assay were 75 min at 65 degrees C, respectively. The LAMP method accurately identified 16 isolates of V. cholerae but did not detect 28 non-cholerae Vibrio isolates and 37 non-Vibrio bacterial isolates. The sensitivity of LAMP for V. cholerae detection in pure cultures was 2.2 x 10(3) CFU ml(-1) or equivalent to 8 CFU per reaction. In the case of spiked shrimp samples without enrichment, the detection limit for V. cholerae was 2.2 x 10(4) CFU g(-1) or equivalent to 20 CFU per reaction, while that of PCR was 100 CFU per reaction.
The developed LAMP assay targeting ompW gene was rapid, specific and sensitive for V. cholerae detection.
The developed LAMP assay appears to be precise, accurate and a valuable tool for detection of V. cholerae. This assay can replace laborious biochemical tests for the identification of V. cholerae in contaminated food sample.
本研究旨在开发一种环介导等温扩增（LAMP）检测方法，用于快速、特异性检测霍乱弧菌。
设计了一组 5 个针对霍乱弧菌 ompW 基因的特异性引物。LAMP 反应的最佳时间和温度条件分别为 75 分钟和 65°C。该 LAMP 方法准确鉴定了 16 株霍乱弧菌，但未检测到 28 株非霍乱弧菌弧菌和 37 株非弧菌细菌分离株。LAMP 法检测纯培养物中霍乱弧菌的灵敏度为 2.2×10³CFU ml（-1）或相当于每个反应 8 个 CFU。在未富集的虾样中添加的情况下，霍乱弧菌的检测限为 2.2×10⁴CFU g（-1）或相当于每个反应 20 个 CFU，而 PCR 的检测限为每个反应 100 CFU。
针对 ompW 基因的开发的 LAMP 检测方法具有快速、特异性和灵敏性，可用于检测霍乱弧菌。
开发的 LAMP 检测方法似乎是一种精确、准确且有价值的霍乱弧菌检测工具。该检测方法可以替代污染食物样本中霍乱弧菌的繁琐生化检测。
