活的非可培养状态副溶血性弧菌实时荧光逆转录PCR检测方法的建立及其毒力研究  

Determination and analysis of potential virulence of viable but nonculturable Vibrio parahaemolyticus

作　　者：刘静宇[1] 凌莉[1] 邓翼惠 易敏英[1] 胡科锋[1] 陈碧玲[1] 

机构地区：[1]广东出入境检验检疫局技术中心,广东广州510623 [2]深圳市龙岗区平湖预防保健所,广东深圳518111

出　　处：《中国食品卫生杂志》2013年第4期309-315,共7页Chinese Journal of Food Hygiene

基　　金：广东省科技计划项目(2011B031500012);广东检验检疫局科技计划项目(2010GDK30)

摘　　要：目的建立检测活的非可培养(VBNC)状态副溶血性弧菌的荧光逆转录PCR方法并研究其毒力基因表达。方法将副溶血性弧菌AS079菌株添加到陈化海水中,4℃冰箱内培养,使其进入VBNC状态,针对其管家基因、鉴定基因和毒力基因分别设计实时荧光逆转录PCR引物,通过不同的PCR反应程序和引物浓度组合试验,摸索最佳反应体系条件,用于VBNC状态副溶血性弧菌的检测和毒力研究。结果用所建立的检测VBNC状态副溶血性弧菌的实时荧光逆转录PCR方法,对接种于陈化海水培养的不同时期的AS079副溶血性弧菌进行荧光定量逆转录PCR扩增,结果显示,随着培养时间的延长,毒力基因tdh2和鉴定基因toxR表达水平持续下降,但即使细菌进入VBNC状态,这两个基因也能够得到很好的扩增,说明以toxR基因和tdh2基因对进入VBNC状态的副溶血性弧菌进行检测和毒力研究方法可行。灵敏度试验表明,鉴定基因toxR的最低检测限可达到48 cfu/ml,研究毒力基因tdh2的表达需要细菌浓度至少为4.8×102cfu/ml,同时试验证明此方法与其他相近食源性致病菌无交叉反应。结论该实时荧光逆转录PCR方法检测快速、特异性强、敏感度高,适用于VBNC状态副溶血性弧菌的检测和毒力分析。Objective To establish a real time RT-PCR method to detect VBNC Vibrio Parahaemolyticus and study the expression of virulence gene.Methods V.parahaemolyticus strain AS079 was induced into VBNC state by culturing in artificial seawater at 4 ℃.Real-time RT-PCR primers were designed for the toxR,pvuA and tdh2 gene.Various PCR protocol and primer concentration combinations were tested to optimize the PCR procedures for VBNC V.parahaemolyticus as well as virulence analysis.V.parahaemolyticus AS079 samples collected after various periods of incubation were analyzed using real-time RT-PCR method established in this study.Results The result indicated that the expression levels of virulence gene tdh2 and identification gene toxR decreased over incubation in artificial seawater.However,both genes were clearly detectable even after the bacteria had entered VBNC state,suggesting that the method could be used for detection and virulence analysis of V.parahaemolyticus under VBNC state.The sensitivity test showed that the detection limit for identification gene toxR was 48 cfu/ml;whereas evaluating the expression of virulence gene tdh2 required 4.8×102 cfu/ml at least.Meanwhile,no cross-reaction with other closely related food-borne pathogens had been found.Conclusion The real-time RT PCR method established in this study had advantages of fast detection,high specificity and sensitivity,and was suitable for detection and virulence analysis of VBNC V.parahaemolyticus.

关 键 词：副溶血性弧菌 活的非可培养 实时荧光逆转录PCR 毒力基因 食源性致病菌 

分 类 号：R155.5[医药卫生—营养与食品卫生学] TS201.6[医药卫生—公共卫生与预防医学]