期刊
FOOD CONTROL
卷 63, 期 -, 页码 230-238出版社
ELSEVIER SCI LTD
DOI: 10.1016/j.foodcont.2015.11.043
关键词
Vibrio parahaemolyticus; Viable but nonculturable state; Real-time fluorescent LAMP; PMA
资金
- National Natural Science Foundation of China [31271956]
- China Scholarship Council
Vibrio parahaemolyticus is a crucial foodborne pathogen. The viable but non-culturable (VBNC) state of V. parahaemolyticus cannot be detected by traditional culture methods. The objective of this study was to develop and evaluate a method that combines propidium monoazide (PMA) treatment with real-time fluorescent loop-mediated isothermal amplification (RF-LAMP) to detect and quantify VBNC cells of V. parahaemolyticus. Different states of cells were treated with PMA in dark for 5 min and subsequently exposed to a 650 W halogen lamp for 5 min. The cells were collected and DNA was amplified by RF-LAMP. The primers which targeted six distinct regions in the tlh gene of V parahaemolyticus were used for the PMA-RF-LAMP assay. The results indicated that the treatment with 4 mu g/mL of PMA and a further exposure to light for 5 min was suitable for PMA-RF-LAMP to distinguish viable cells from dead cells of V parahaemolyticus. The developed assay exhibited remarkably high specificity, sensitivity and rapidity, without any cross reaction with the tested non-V. parahaemolyticus strains. There was good linear correlation between Ct values and log copy/mL in the range of 6.8-6.8 x 10(7) copy/mL, with the reaction endpoints less than 30 cycles (30 min). It could detect as low as 14 copy/g of V parahaemolyticus in spiked seafood samples (pomfret, shrimp, scallop, oyster and salted fish) without any interference from food matrices, dead cells and other bacteria. (C) 2015 Elsevier Ltd. All rights reserved.
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