Department of Genetic Engineering, Faculty of Biosciences and Biotechnology, Malekashtar University of Technology, Tehran, Iran.
J Infect Public Health. 2012 Jun;5(3):263-7. doi: 10.1016/j.jiph.2012.02.004. Epub 2012 May 1.
Vibrio cholerae (V. cholerae) is the etiological agent of epidemic cholera. In recent years, cholera has become endemic in different regions of the world. Traditional culture and microscopic methods are considered the standard for the diagnosis of V. cholerae. However, these methods require days for confirmation. Therefore, molecular methods that may be used for the sensitive, accurate, and rapid analysis of V. cholerae are urgently needed.
In the present investigation, a multiplex PCR assay was developed for the detection of virulence- and toxigenic-associated (VTA) genes (ctxA, tcpA and ompW). To evaluate PCR specificity, other bacteria from the Enterobacteriaceae family (Salmonella typhi, Shigella dysenteriae, and enterotoxigenic Escherichia coli) and Aeromonas hydrophila were examined. The assay sensitivity was evaluated using colony counting and genome dilution methods.
Our results showed that this PCR-based method represents an ideal tool for the rapid detection of VTA genes due to its simplicity, cost effectiveness, and accuracy. This multiplex PCR method can be used to determine the presence of VTA genes and can therefore distinguish V. cholerae bacteria from other Vibrio species and bacteria. This method can detect 10-100 CFU V. cholerae and 8.5-85 pg genomic DNA.
This multiplex PCR method has higher sensitivity and specificity than other methods. The proposed test provides an appropriate and sensitive tool for detecting the presence of toxigenic and pathogenic V. cholerae.
霍乱弧菌（V. cholerae）是霍乱的病原体。近年来，霍乱在世界不同地区已成为地方病。传统的培养和显微镜方法被认为是诊断 V. cholerae 的标准方法。然而，这些方法需要数天才能得到确认。因此，迫切需要能够用于敏感、准确和快速分析 V. cholerae 的分子方法。
在本研究中，开发了一种用于检测毒力和产毒相关（VTA）基因（ctxA、tcpA 和 ompW）的多重 PCR 检测方法。为了评估 PCR 的特异性，还检测了肠杆菌科（伤寒沙门氏菌、志贺氏菌和肠产毒性大肠杆菌）和嗜水气单胞菌中的其他细菌。通过菌落计数和基因组稀释方法评估了检测方法的灵敏度。
我们的结果表明，由于该基于 PCR 的方法简单、经济有效且准确，因此是一种快速检测 VTA 基因的理想工具。这种多重 PCR 方法可用于确定 VTA 基因的存在，因此可以区分霍乱弧菌和其他弧菌属和细菌。该方法可检测到 10-100 CFU 的 V. cholerae 和 8.5-85 pg 基因组 DNA。
这种多重 PCR 方法比其他方法具有更高的灵敏度和特异性。该检测方法为检测产毒和致病 V. cholerae 提供了一种合适且敏感的工具。
