不同诱导条件下的VBNC状态副溶血弧菌的PMA-qPCR定量检测及其呼吸活性分析

摘要

The aims of the study were to quantitatively monitor viable but non-culturable (VBNC) cells of Vibrio parahaemolyticus induced by different conditions and to analyze their respiratory activity.Fluorescence quantitative PCR (qPCR) coupled with propidium monoazide (PMA-qPCR) was used to quantify viable cells of V.parahaemolyticus.A new method combining confocal laser-scanning microscopy (CLSM) and flow cytometry with 5-cyano-2,3-ditolyl tetrazolium chloride (CTC) reduction was applied to detect the respiratory activity of VBNC cells.Results showed that PMA-qPCR with culture-based method allowed successful monitoring of the number of VBNC cells of V.parahaemolyticus and then the density of VBNC cells could be calculated.Seven of the tested conditions could entirely induce cells into VBNC state within 60 day.The minimum and maximum density of VBNC cells were 5.75 × 102 and 1.70 × 105 CFU/mL,respectively.In the respiration test,5-cyano-2,3-ditolyl tetrazolium chloride/4',6-diamidino-2-phenylindole (CTC/ DAPI) staining allowed to clearly distinguish viable cells from dead cells under CLSM by fluorescent formazan.Flow cytometry enabled rapid discrimination between respiratory active and non-active cells based on fluorescence intensity.In summary,this study provided new methods for the detection of V.parahaemolyticus in VBNC state and for better understanding of their metabolism.%定量检测不同诱导条件下的活的非可培养(viable but non-culturable,VBNC)状态副溶血弧菌的变化情况,并对VBNC菌的呼吸活性进行分析.采用叠氮溴化丙锭(propidium monoazide,PMA)与荧光定量聚合酶链式反应(fluorescence quantitative polymerase chain reaction,qPCR)结合的技术(PMA-qPCR)定量检测副溶血弧菌活菌数.结合激光扫描共聚焦显微镜和流式细胞仪优化传统CTC(5-cyano-2,3-ditolyl tetrazolium chloride)呼吸检测法对VBNC状态的副溶血弧菌进行分析.结果表明PMA-qPCR检测技术结合平板计数的方法,可用于定量检测副溶血弧菌诱导过程中活菌数和可培养菌数的变化情况,并利用差值测定出VBNC细胞的浓度.在不同的温度、NaC1含量和氯霉素含量的诱导条件下,副溶血弧菌在其中的7种条件下在60 d内进入了VBNC状态.VBNC细胞终浓度最低为5.75×102 CFU/mL,最高为1.70×105 CFU/mL.在细胞呼吸实验中,采用CTC与4',6-二脒基-2-苯基吲哚(4',6-diamidino-2-phenylindole,DAPI)双染法能在激光扫描共聚焦显微镜下清晰地观察到VBNC细胞表面的红色荧光,有效区分死活细胞.同时利用流式细胞仪能够根据荧光强度快速区分呼吸活性呈阴性和阳性的细胞.本研究为VBNC细菌的检测和生理特性的研究提供了新的思路和依据.