Rapid method for species-specific identification of Vibrio cholerae using primers targeted to the gene of outer membrane protein OmpW
Rapid method for species-specific identification of Vibrio cholerae using primers targeted to the gene of outer membrane protein OmpW
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DOI：
10.1128/jcm.38.11.4145-4151.2000
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发表时间：
2000-11-01
影响因子：
9.4
通讯作者：
Ghose, AC
中科院分区：
文献类型：
--
作者：
Nandi, B;Nandy, RK;Ghose, AC
使用相应的引物和探针研究了霍乱弧菌和其他生物体中的外膜蛋白（OmpW）和调节蛋白（ToxR）的基因分布。PCR扩增结果显示254株霍乱弧菌ompW均为阳性（100%），233株霍乱弧菌toxR阳性229株（98%）。40株属于其他弧菌属的菌株中没有一株用ompW或toxR特异性引物产生扩增子，而来自其他属的80株细菌菌株也被检测为阴性。在DNA斑点印迹试验中，使用ompW和toxR特异性探针对代表性数量的菌株进行了扩展。虽然霍乱弧菌菌株与ompW探针反应，但测试的60种其他细菌菌株中只有一种（拟态弧菌）显示出弱识别。相反，属于其他弧菌物种的几种菌株（例如，拟态弧菌、华丽弧菌、溶藻弧菌、河流弧菌、溶蛋白弧菌、河口弧菌、Isalmonidida、V. pagissii和V. parahaemolyticus）对toxR探针显示出弱至强的反应性。限制性片段长度多态性分析和核苷酸序列数据显示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。这些结果表明ompW基因可以作为霍乱弧菌种特异性鉴定的靶基因。本研究的范围进一步扩大，通过一步多重PCR检测ompW和ctxA基因的同时扩增的发展，这应该是相当大的价值，在临床和环境来源的致病性和非致病性霍乱弧菌菌株的筛选。
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 (similar to 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., lsalmonidida, 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.