Rivera Irma N G, Lipp Erin K, Gil Ana, Choopun Nipa, Huq Anwar, Colwell Rita R
Center of Marine Biotechnology, University of Maryland Biotechnology Institute, Baltimore, MD 21202, USA.
Environ Microbiol. 2003 Jul;5(7):599-606. doi: 10.1046/j.1462-2920.2003.00443.x.
Vibrio cholerae is a free-living bacterium found in water and in association with plankton. V. cholerae non-O1/non-O139 strains are frequently isolated from aquatic ecosystems worldwide. Less frequently isolated are V. cholerae O1 and V. cholerae O139, the aetiological agents of cholera. These strains have two main virulence-associated factors, cholera toxin (CT) and toxin co-regulated pilus (TCP). By extracting total DNA from aquatic samples, the presence of pathogenic strains can be determined quickly and used to improve a microbiological risk assessment for cholera in coastal areas. Some methods suggested for DNA extraction from water samples are not applicable to all water types. We describe here a method for DNA extraction from coastal water and a multiplex polymerase chain reaction (PCR) for O1 and O139 serogroups. DNA extraction was successfully accomplished from 117 sea water samples collected from coastal areas of Perú, Brazil and the USA. DNA concentration in all samples varied from 20 ng to 480 micro g micro l-1. The sensitivity of the DNA extraction method was 100 V. cholerae cells in 250 ml of water. The specificity of multiplex O1/O139 PCR was investigated by analysing 120 strains of V. cholerae, Vibrio and other Bacteria species. All V. cholerae O1 and O139 tested were positive. For cholera surveillance of aquatic environments and ballast water, total DNA extraction, followed by V. cholerae PCR, and O1/O139 serogroup and tcpA/ctxA genes by multiplex PCR offers an efficient system, permitting risk analysis for cholera in coastal areas.
霍乱弧菌是一种自由生活的细菌，存在于水中并与浮游生物有关。霍乱弧菌非O1/非O139菌株在世界各地的水生生态系统中经常被分离出来。霍乱的病原体霍乱弧菌O1和霍乱弧菌O139则较少被分离到。这些菌株有两个主要的毒力相关因子，即霍乱毒素（CT）和毒素共调节菌毛（TCP）。通过从水生样本中提取总DNA，可以快速确定致病菌株的存在，并用于改进沿海地区霍乱的微生物风险评估。一些建议用于从水样中提取DNA的方法并不适用于所有类型的水。我们在此描述一种从沿海水提取DNA的方法以及针对O1和O139血清群的多重聚合酶链反应（PCR）。从秘鲁、巴西和美国沿海地区采集的117份海水样本中成功完成了DNA提取。所有样本中的DNA浓度在20纳克至480微克/微升之间变化。DNA提取方法的灵敏度为250毫升水中100个霍乱弧菌细胞。通过分析120株霍乱弧菌、弧菌属和其他细菌物种，研究了多重O1/O139 PCR的特异性。所有检测的霍乱弧菌O1和O139均为阳性。对于水生环境和压载水的霍乱监测，先进行总DNA提取，然后进行霍乱弧菌PCR，以及通过多重PCR检测O1/O139血清群和tcpA/ctxA基因，提供了一个高效的系统，可用于沿海地区霍乱的风险分析。
