Lee C, Pan S F
Graduate Institute of Agricultural Chemistry, National Taiwan University, Taipei, Republic of China.
J Gen Microbiol. 1993 Dec;139(12):3225-31. doi: 10.1099/00221287-139-12-3225.
Synthetic oligonucleotide primers derived from a sequence of the thermostable direct haemolysin (tdh) gene were used in a polymerase chain reaction (PCR) amplification technique to detect this gene in strains of Vibrio parahaemolyticus. A total of 36 TDH-producing, and 89 TDH-negative Vibrio parahaemolyticus strains and 46 other vibrios and enteric pathogens were studied. In all, 36 strains of Vibrio parahaemolyticus from which the tdh gene could be successfully amplified by PCR were found to be TDH-positive in TDH haemolysin assay. No amplification products were obtained from Vibrio parahaemolyticus strains that were TDH-negative in the haemolysin assay or from other vibrios and enteric pathogens, with the exception of two strains. The PCR results were consistent with DNA hybridization tests. The detection limit for the tdh gene by PCR amplification was 40 pg of total DNA, or broth culture containing 1000 viable cells. Amplification products were confirmed by restriction enzyme digestion and Southern blot hybridization. The PCR method could detect the tdh sequences in stool samples from patients with gastroenteritis caused by V. parahaemolyticus. This PCR protocol clearly identified TDH-producing strains of V. parahaemolyticus and provides an alternative to conventional methods for TDH detection by research laboratories, clinical laboratories, regulatory agencies, and the seafood industry.
从耐热直接溶血素（tdh）基因序列衍生的合成寡核苷酸引物，用于聚合酶链反应（PCR）扩增技术，以检测副溶血性弧菌菌株中的该基因。总共研究了36株产TDH的副溶血性弧菌菌株、89株TDH阴性副溶血性弧菌菌株以及46株其他弧菌和肠道病原体。通过PCR能够成功扩增出tdh基因的36株副溶血性弧菌，在TDH溶血素检测中均为TDH阳性。除两株外，溶血素检测为TDH阴性的副溶血性弧菌菌株以及其他弧菌和肠道病原体均未获得扩增产物。PCR结果与DNA杂交试验一致。通过PCR扩增检测tdh基因的检测限为40 pg总DNA或含有1000个活细胞的肉汤培养物。扩增产物通过限制性内切酶消化和Southern印迹杂交进行确认。PCR方法能够检测副溶血性弧菌引起的肠胃炎患者粪便样本中的tdh序列。该PCR方案能够清晰鉴定产TDH的副溶血性弧菌菌株，为研究实验室、临床实验室、监管机构和海产品行业提供了一种替代传统TDH检测方法的手段。
