PMA based real-time fluorescent LAMP for detection of Vibrio parahaemolyticus in viable but nonculturable state |
| |
| Affiliation: | 1. College of Food Science, South China Agricultural University, Guangzhou 510642, China;2. Guangzhou South China Biological Medicine Co. Ltd., Guangzhou 511300, China;1. Guangdong Inspection and Quarantine Technology Center, Microbiological Test Department of Food Testing Lab, 510623 Guangzhou, PR China;2. College of Light Industry and Food Sciences, South China University of Technology, 510640 Guangzhou, PR China;3. Graduate School of Life and Environment Sciences, Osaka Prefecture University, Osaka 598-8531, Japan;4. International Centre for Diarrhoeal Diseases Research, Mohakhali, Dhaka 1212, Bangladesh;5. Guangzhou DE AOU Biotechnology Technology Co., Ltd, Innovation Building, Science Road, Guangzhou Science City, 510663 Guangzhou, PR China;1. College of Food Science and Engineering, Northwest A&F University, Yangling, Shaanxi 712100, PR China;2. Key Laboratory of Bio-environmental Engineering, Ministry of Agriculture, China Agricultural University, P.O. Box 67, Beijing 100083, PR China;3. Department of Food Science and Technology, University of Georgia, 1109 Experiment Street, Griffin, GA 30223, USA |
| |
| Abstract: | 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 μg/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 × 107 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. |
| |
| Keywords: | Viable but nonculturable state Real-time fluorescent LAMP PMA |
| 本文献已被 ScienceDirect 等数据库收录！ |
|
