食品科学2017，Vol.38Issue(20)：215-221,7.DOI:10.7506/spkx1002-6630-201720031
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
Quantitative Detection of Viable but Non-Culturable (VBNC) Vibrio parahaemolyticus Cells Induced by Different Conditions Using PMA-qPCR and Respiratory Activity Analysis
摘要
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.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)分类
轻工纺织引用本文复制引用
刘羽霏,方祥,廖振林,王丽,钟青萍..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[J].食品科学,2017,38(20):215-221,7.基金项目
国家自然科学基金面上项目(31271956) （31271956）
广东省自然科学基金项目(2017A030313146) （2017A030313146）
