Nandi B, Nandy R K, Mukhopadhyay S, Nair G B, Shimada T, Ghose A C
Department of Microbiology, Bose Institute, Calcutta 700 054, India.
J Clin Microbiol. 2000 Nov;38(11):4145-51. doi: 10.1128/JCM.38.11.4145-4151.2000.
The distribution of genes for an outer membrane protein (OmpW) and a regulatory protein (ToxR) in Vibrio cholerae and other organisms was studied using respective primers and probes. PCR amplification results showed that all (100%) of the 254 V. cholerae strains tested were positive for ompW and 229 ( approximately 98%) of 233 were positive for toxR. None of the 40 strains belonging to other Vibrio species produced amplicons with either ompW- or toxR-specific primers, while 80 bacterial strains from other genera tested were also found to be negative by the assay. These studies were extended with representative number of strains using ompW- and toxR-specific probes in DNA dot blot assay. While the V. cholerae strains reacted with ompW probe, only one (V. mimicus) out of 60 other bacterial strains tested showed weak recognition. In contrast, several strains belonging to other Vibrio species (e.g., V. mimicus, V. splendidus, V. alginolyticus, V. fluvialis, V. proteolyticus, V. aestuarianus, V. salmonicida, V. furnissii, and V. parahaemolyticus) showed weak to strong reactivity to the toxR probe. Restriction fragment length polymorphism analysis and nucleotide sequence data revealed that the ompW sequence is highly conserved among V. cholerae strains belonging to different biotypes and/or serogroups. All of these results suggest that the ompW gene can be targeted for the species-specific identification of V. cholerae strains. The scope of this study was further extended through the development of a one-step multiplex PCR assay for the simultaneous amplification of ompW and ctxA genes which should be of considerable value in the screening of both toxigenic and nontoxigenic V. cholerae strains of clinical as well as environmental origin.
使用各自的引物和探针研究了霍乱弧菌及其他生物中外膜蛋白（OmpW）基因和调节蛋白（ToxR）基因的分布情况。PCR扩增结果显示，所检测的254株霍乱弧菌全部（100%）ompW呈阳性，233株中有229株（约98%）toxR呈阳性。属于其他弧菌属的40株菌株中，没有一株能用ompW或toxR特异性引物产生扩增子，同时检测的来自其他属的80株细菌菌株经该检测也呈阴性。使用ompW和toxR特异性探针，通过DNA斑点杂交试验，以具有代表性数量的菌株扩展了这些研究。虽然霍乱弧菌菌株与ompW探针发生反应，但所检测的60株其他细菌菌株中只有一株（模仿弧菌）显示出微弱的识别反应。相反，几种属于其他弧菌属的菌株（如模仿弧菌、灿烂弧菌、溶藻弧菌、河流弧菌、解蛋白弧菌、河口弧菌、杀鲑弧菌、弗尼斯弧菌和副溶血弧菌）对toxR探针显示出弱到强的反应性。限制性片段长度多态性分析和核苷酸序列数据表明，ompW序列在属于不同生物型和/或血清群的霍乱弧菌菌株中高度保守。所有这些结果表明，ompW基因可用于霍乱弧菌菌株的种特异性鉴定。通过开发一步多重PCR检测法同时扩增ompW和ctxA基因，进一步扩展了本研究的范围，这在筛查临床和环境来源的产毒和不产毒霍乱弧菌菌株方面应具有相当大的价值。
