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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 fluorescence reverse transcription-PCR method to detect Vibrio parahaemolyticus (Vibrio parahaemolyticus) in live non-cultureable (VBNC) state and study its virulence gene expression. Methods The Vibrio parahaemolyticus strain AS079 was added into aged seawater and cultured in refrigerator at 4 ℃ to bring it into VBNC state. Real-time fluorescent reverse transcription PCR primers were designed according to their housekeeping genes, identification genes and virulence genes respectively. PCR reaction program and primer concentration combination test, explore the best reaction system conditions, for the detection of VBNC status of Vibrio parahaemolyticus and virulence studies. Results Real-time fluorescence reverse transcription PCR (RT-PCR) was used to detect Vibrio parahaemolyticus in VBNC. Fluorescence quantitative reverse transcriptase-PCR (RT-PCR) was used to amplify Vibrio parahaemolyticus AS079 inoculated in aged seawater. As the incubation time prolonged, the expression level of tdh2 and toxR of the virulence genes continued to decline, but even the bacteria entered the VBNC state, these two genes could be well amplified, indicating that toxR gene and tdh2 gene pair enter VBNC Vibrio parahaemolyticus state of detection and virulence research methods feasible. The sensitivity test showed that the detection limit of toxR was 48 cfu / ml. To study the expression of tdh2, the bacterial concentration should be at least 4.8 × 102 cfu / ml. At the same time, this method was compared with other foodborne pathogens No cross-reaction. Conclusion The real-time fluorescence reverse transcription PCR method is rapid, specific and sensitive and suitable for the detection and virulence analysis of Vibrio parahaemolyticus in VBNC.