基于环介导等温扩增法（LAMP）检测上海市售贝类产品中副溶血性弧菌的毒力菌株

Detection of Virulent Vibrio parahaemolyticus Strains from Commercial Shellfish in Shanghai by Loop-mediated Isothermal Amplification

摘要:

基于环介导等温扩增法（LAMP）对上海市8-10月市售贝类产品中副溶血性弧菌毒力菌株（tdh和trh毒力基因）进行检测分析，共检测贝类样品180份，6个常规品种，实验同时采用PCR测定方法进行对比。结果表明，含tdh和trh毒力基因的副溶血性弧菌在市售贝类中的检出率分别是12.77%和11.66%，PCR的分析结果为11.11%和7.78%。对分离的毒力菌株进行血清型分型后发现了2株O3:K6型副溶血性弧菌，其中1株为毒力基因双阳性菌（tdh+/ trh+）。2株O3:K6型副溶血性弧菌的PFGE条带型相似度较高（相似度>90%）。这些结果表明上海市售贝类产品中副溶血性弧菌毒力菌株存在一定的污染，应引起足够重视。双阳性O3:K6型副溶血性弧菌的出现值得关注，应对各血清型菌株尤其是O3:K6型副溶血性弧菌的流行情况加强监测。PCR检测结果对比分析表明，LAMP方法适用于贝类产品中副溶血性弧菌毒力菌株的检测分析。

Abstract:

Loop-mediated isothermal amplification (LAMP) was used to detect and analyze virulence-associated genes, thermostable direct hemolysin (tdh) and thermostable-related hemolysin (trh), in Vibrio parahaemolyticus strains isolated from commercial shellfish in Shanghai from August to October, 2014. A total of 180 samples of six common species of shellfish were studied and results were compared between LAMP and polymerase chain reaction (PCR) assays. The detection rates of tdh and trh genes in Vibrio parahaemolyticus strains from commercial shellfish were 12.77% and 11.66% using LAMP as compared to 10.55% and 7.77% by PCR, respectively. Serotyping the isolated virulent strains revealed two O3: K6 serotype Vibrio parahaemolyticus strains, including one carrying double-positive virulence genes (tdh+/trh+). Additionally, high similarity was observed between pulsed-field gel electrophoresis (PFGE) bands of two O3: K6 serotype Vibrio parahaemolyticus strains (similarity > 90%). The results indicate contamination of commercial shellfish in Shanghai by virulent strains of Vibrio parahaemolyticus, where the emergence of tdh+/trh+ O3:K6 serotype Vibrio parahaemolyticus is a cause for concern. Surveillance needs to be strengthened, for Vibrio parahaemolyticus strains of different serotypes, especially the O3: K6 serotype. Results from the comparison with PCR assay indicated that LAMP method was suitable for the detection of virulent strains of Vibrio parahaemolyticus in shellfish.