School of Biomedical and Biological Sciences, University of Plymouth, Plymouth, PL4 8AA, UK.
Eur J Clin Microbiol Infect Dis. 2012 Apr;31(4):431-9. doi: 10.1007/s10096-011-1324-9. Epub 2011 Jul 3.
Vibrio parahaemolyticus is a marine bacterium and some strains cause gastroenteritis in humans. Clinical isolates are thought to possess virulence factors that are absent from the majority of environmental isolates. Use of randomly amplified polymorphic DNA (RAPD)-PCR produced a unique 600 bp amplicon (band Y) in the majority of clinical isolates and rarely in environmental isolates tested. The DNA from band Y was cloned and sequenced and found to code for an outer membrane protein (OMP). Two polymerase chain reaction (PCR) primers were designed to specifically amplify a 200 bp unique sequence from presumptive virulent strains (PCR-OMP). The virulence of 23 clinical and 32 environmental isolates was assessed in cytotoxicity tests by treatment of Caco-2 cells with extracellular products (ECPs). All but two of the clinical isolates (91%) were positive for the 200 bp PCR-OMP and their ECPs produced a significantly higher (p < 0.05) lactate dehydrogenase (LDH) release (mean 72.88%) than the ECPs of environmental isolates (mean 15.3%) with the exception of one environmental isolate that produced the 200 bp amplicon. A positive 200 bp PCR-OMP is strongly correlated with virulence, as determined by the cytotoxicity assay, and identified virulent forms better than current PCR tests for tdh, trh or T3SS2.
副溶血性弧菌是一种海洋细菌，其中一些菌株会导致人类患肠胃炎。临床分离株被认为具有毒力因子，而这些毒力因子在大多数环境分离株中不存在。使用随机扩增多态性 DNA (RAPD)-PCR 在大多数临床分离株中产生了一个独特的 600 bp 扩增子（带 Y），而在测试的环境分离株中很少出现。带 Y 的 DNA 被克隆和测序，发现它编码一种外膜蛋白 (OMP)。设计了两个聚合酶链反应 (PCR) 引物，用于特异性扩增推定毒力株的 200 bp 独特序列（PCR-OMP）。通过用细胞外产物 (ECP) 处理 Caco-2 细胞，在细胞毒性试验中评估了 23 株临床分离株和 32 株环境分离株的毒力。除了两个临床分离株（91%）外，所有临床分离株都对 200 bp PCR-OMP 呈阳性，其 ECP 产生的乳酸脱氢酶 (LDH) 释放量（平均 72.88%）明显高于环境分离株（平均 15.3%），除了一个产生 200 bp 扩增子的环境分离株。阳性 200 bp PCR-OMP 与细胞毒性测定确定的毒力呈强相关性，并且比当前用于 tdh、trh 或 T3SS2 的 PCR 检测更好地识别毒力形式。
