Département Ressources Biologiques et Environnement, Unité Environnement Microbiologie et Phycotoxines, Ifremer, Centre de Brest, ZI de la Pointe du Diable, Plouzané, France.
J Appl Microbiol. 2012 Aug;113(2):361-72. doi: 10.1111/j.1365-2672.2012.05350.x.
To identify Vibrio vulnificus, Vibrio cholerae and Vibrio alginolyticus using standardized DNA extraction method and real-time PCR assays, among a large number of bacterial strains isolated from marine environment.
Methods for DNA extraction and real-time PCR were standardized to identify a large number of Vibrio spp. strains isolated through regular collection campaigns of environmental samples. Three real-time PCR assays were developed from a multiplex PCR, targeting V. vulnificus, V. cholerae and V. alginolyticus on the dnaJ gene. After testing their specificity, these systems were applied for the identification of 961 strains isolated at 22°C (446 strains) and 37°C (515 strains) in September 2009. The predominance of V. alginolyticus (82·6%) among the Vibrio spp. strains isolated at 37°C was shown. At 22°C, only 1·6% of the strains were identified by PCR and they were V. alginolyticus.
Reproducible and specific real-time PCR assays combined to a DNA extraction method on microplates were used to constitute a large environmental Vibrio strains collection and to identify and detect potential human pathogenic Vibrio isolated at 37°C. For environmental strains isolated at 22°C, because of the higher species diversity, other approaches, like sequencing, should be chosen for identification.
The protocol developed in this study provides an appropriate and rapid screening tool to identify a large number of bacterial strains routinely isolated from the environment in long-term studies.
使用标准化 DNA 提取方法和实时 PCR 检测，从大量海洋环境中分离的细菌菌株中鉴定创伤弧菌、霍乱弧菌和溶藻弧菌。
方法标准化了 DNA 提取和实时 PCR，以鉴定通过定期采集环境样本进行的大量弧菌属菌株。从多重 PCR 中开发了 3 种针对 dnaJ 基因的实时 PCR 检测方法，用于鉴定创伤弧菌、霍乱弧菌和溶藻弧菌。在测试其特异性后，将这些系统应用于 2009 年 9 月在 22°C（446 株）和 37°C（515 株）下分离的 961 株菌株的鉴定。结果表明，在 37°C 下分离的弧菌属菌株中，溶藻弧菌（82.6%）占优势。在 22°C 下，只有 1.6%的菌株通过 PCR 鉴定为溶藻弧菌。
可重复且特异的实时 PCR 检测与微孔板上的 DNA 提取方法相结合，构成了大量环境弧菌菌株的采集，并鉴定和检测了在 37°C 下分离的潜在人类致病弧菌。对于在 22°C 下分离的环境菌株，由于物种多样性较高，应选择测序等其他方法进行鉴定。
本研究中开发的方案为从环境中常规分离的大量细菌菌株提供了一种合适且快速的筛选工具，可用于长期研究。
