巢式PCR快速检测海产品中的副溶血弧菌

Rapid Identification of Vibrio parahaemolyticus in Seafood by Nested PCR

摘要:

副溶血弧菌是一种世界范围性的食源性致病菌，食用了该菌污染的海产品可导致胃肠炎等疾病。为了建立一种可快速、特异地检测海产品中副溶血弧菌的方法，通过把副溶血弧菌基因组序列和其它不同种类弧菌的基因组序列进行比较分析，筛选出了一个副溶血弧菌特异性的标记基因-VP1331，根据该基因建立了副溶血弧菌的巢式PCR快速检测方法，并评估了其特异性、敏感性和稳定性。实验结果表明，该方法只有在以副溶血弧菌基因组DNA为模板时才能扩增出目的片段，而其它11种弧菌和非弧菌均不能扩增出目的片段。该方法的最低检测限为副溶血弧菌基因组DNA 10 fg、纯培养物6.6 CFU。人工污染实验表明，初始菌液浓度为25.7 CFU/100 mL时只需经过2 h的增菌培养即可检出。上述结果表明，VP1331基因可以作为副溶血弧菌种特异性标记，本方法可以用于污染海产品中该菌的检测与鉴定。

Abstract:

Vibrio parahaemolyticus is a food-borne pathogen that causes gastroenteritis and other diseases worldwide, with consumption of contaminated raw or undercooked seafood. To develop a method for rapid and specific detection of V. parahaemolyticus in seafood, its genomic sequence was analyzed and compared with those of other Vibrio species, a V. parahaemolyticus-specific genetic marker was screened, and a nested-PCR-based method to rapid detect V. parahaemolyticus was established based on this gene. Additionally, the specificity, sensitivity, and reproducibility of this method were assessed. The results showed that the target amplicon only appeared with V. parahaemolyticus genomic DNA as the template, but not the other 11 Vibrio species and non-Vibrio species. The limits of detection of this method were 10 fg V. parahaemolyticus genomic DNA and 6.6 colony-forming units (CFU) for purified culture. Artificial contamination experiments indicated that V. parahaemolyticus could be detected after two hours of enrichment with an initial concentration of 25.7 CFU/100 mL. In conclusion, the VP1331 gene can be used as a V. parahaemolyticus-specific marker, and this PCR method can be used for the detection and identification of V. parahaemolyticus in contaminated seafood.