关键词: 副溶血弧菌, 活的非可培养（VBNC）状态, PMA-qPCR, 流式细胞仪, 激光扫描共聚焦显微镜

Abstract: 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.

Key words: Vibrio parahaemolyticus, viable but non-culturable(VBNC), fluorescence quantitative PCR combined with propidium monoazide (PMA-qPCR), flow cytometry, confocal laser-scanning microscopy (CLSM)