海鲜产品副溶血弧菌增菌培养前后tdh^-/tdh^+菌株比例差异分析  

Analysis of the difference in the ratio of tdh^-/tdh^+strains before and after enrichment of Vibrio parah-aemolyticus in retail seafood

作　　者：鞠长燕[1] 马艳萍 刘敏 段永翔[1] 逄波[2] 张茂俊[2] JU Chang-yan;MA Yan-ping;LIU Min;DUAN Yong-xiang;PANG Bo;ZHANG Mao-jun(Nanshan Center for Disease Control and Prevention of Shenzhen,Shenzhen,China 518054;Chinese Center for Disease Control and Prevention)

机构地区：[1]深圳市南山区疾病预防控制中心,广东深圳518051 [2]中国疾病预防控制中心传染病预防控制所

出　　处：《中国病原生物学杂志》2020年第4期402-405,共4页Journal of Pathogen Biology

基　　金：深圳市科技创新委员会项目(No.JCYJ20160427110027326);深圳市"医疗卫生三名工程"引进高层次医学团队项目(No.SZSM201803081);深圳市南山区重点学科建设项目。

摘　　要：目的分析海鲜产品中副溶血弧菌tdh^-/tdh^+株在增菌培养前后的比例差异,探讨副溶血弧菌相关食源性疾病中tdh^+菌株在食品中难以检测到的原因。方法采集市场零售的50份海鲜样品,根据GB 4789.7-2013进行副溶血弧菌的增菌培养。采用双重荧光定量PCR,利用标准曲线确定tdh^-和tdh^+菌株的分布比例。采用模拟试验,将6组tdh^-/tdh^+株配对,在总菌量为10^3、10^4、10^5、10^6cfu/mL条件下混合增菌培养,测定增菌前后tdh^-/tdh^+株的比例。绘制上述12株副溶血弧菌的单菌生长曲线,进一步比对tdh-和tdh^+株的生长速度。结果荧光PCR显示,50份海鲜产品中副溶血弧菌的污染范围为2.1×10^4^5.1×10^6cfu/g,增菌前tdh^-/tdh^+株的比例范围为2.2×10^2∶1^1.9×10^5∶1,增菌后tdh^-/tdh^+株的比例范围为7.1×10^2∶1^3.3×10^5∶1。混合生长试验显示,tdh^-/tdh^+株初始比例范围为1.8∶1^85∶1,增菌后tdh^-/tdh^+株比例范围变为0.1∶1^11∶1。总体上,初始菌量接近的情况下进行混合培养,tdh^+株的生长速度并不比tdh^-株低。单菌生长曲线显示,tdh^-株与tdh^+株在相同条件下生长速度无明显差异。结论与tdh^-株相比,海鲜产品中tdh^+株所占比例低,增菌后所占比例更低,导致tdh^+株在海鲜产品样本增菌后难以被检出,而非tdh^+株本身的生长速度低于tdh-株造成。Objective To analyze the ratio of tdh^-/tdh^+strains of Vibrio parahaemolyticus in seafood samples before and after enrichment and to explore the possible reasons why detecting tdh^+strains in food samples is difficult during outbreaks of foodborne diseases related to V.parahaemolyticus.Methods Fifty seafood samples were collected from retail markets,and V.parahaemolyticus was enriched in accordance with GB 4789.7-2013.Duplex real-time PCR was used to determine the ratio of tdh^-/tdh^+strains before and after enrichment.Six tdh^-strains and 6 tdh^+strains were paired and co-cultured to reach 103,104,105,or 106 CFU/mL of total bacteria,and the ratio of tdh^-/tdh^+strains was also determined before and after enrichment.The growth rates of 6 tdh^-strains and 6 tdh^+strains were compared by growth curves.Results The prevalence of V.parahaemolyticus in seafood was 96%(48/50)in this study,and the concentration of V.parahaemolyticus ranged from 2.1×10^4 to 5.1×10^6 CFU/g before enrichment.The ratio of tdh^-/tdh^+strains was 2.2×10^2:1-1.9×10^5:1initially and then became 7.1×10^2:1to 3.3×10^5:1after enrichment.In co-cultured broth,the initial ratio of tdh^-/tdh^+strains was 1.8:1-85:1,which became 0.1:1-11:1after enrichment.Generally,the growth rate of tdh^+strains was not lower than that of tdh^-strains when the initial ratio was close.The growth curve indicated that V.parahaemolyticus grew fast in the logarithmic period of 0-20000sand gradually entered the stationary period after 30000s.However,there was no significant difference between the growth rate of tdh^-and tdh^+strains.Conclusion There were fewer tdh^+strains than tdh^-strains in seafood,and that ratio further declined after enrichment.As a result,tdh^+strains could not be readily isolated in seafood samples.The growth rate of tdh^+strains was lower than that of tdh^-strains.

关 键 词：副溶血弧菌 耐热直接溶血素 荧光定量PCR 生长曲线 

分 类 号：R378.3[医药卫生—病原生物学]