Real time loop-mediated isothermal amplification using a portable fluorescence scanner for rapid and simple detection of Vibrio parahaemolyticus |
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| Affiliation: | 1. East China Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences, Key Laboratory of East China Sea and Oceanic Fishery Resources Exploitation and Utilization, Ministry of Agriculture, Shanghai 200090, China;2. Beijing Advanced Innovation Center for Food Nutrition and Human Health, College of Veterinary Medicine, China Agricultural University, Beijing, China;3. Advanced Institute of Translational Medicine, Tongji University, Shanghai 200092, China;1. Istituto Superiore di Sanità, Department of Veterinary Public Health and Food Safety, viale Regina Elena 299, 00161 Rome, Italy;2. Istituto Zooprofilattico Sperimentale delle Venezie, viale Università 10, 35020 Legnaro, PD, Italy;3. University of Sassari, Department of Veterinary Medicine, via Vienna 2, 07100 Sassari, Italy;4. University of Bologna, Department of Veterinary Medical Sciences, viale Vespucci 2, 47042 Cesenatico, FC, Italy |
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| Abstract: | We evaluated the usefulness of the loop-mediated isothermal amplification (LAMP) using a portable ESE Quant tube scanner as a rapid and simple method for the detection of Vibrio parahaemolyticus, an important pathogen causing seafood-borne gastroenteritis. The real time LAMP (RT-LAMP) assay using a hemolysin gene (tlh/ldh)-specific primers was verified using V. parahaemolyticus strains (n = 91) from different countries and other non-target strains to check the utility of this method. Both the sensitivity and specificity of the RT-LAMP using 3 pairs of tlh/ldh-specific primers developed in this study were excellent (100%). The detection limit of the RT-LAMP was as low as 7 Colony forming unit per reaction and detection time was only 20 min. Comparative evaluation of the target bacterial strains with the RT-LAMP using ESE Quant tube scanner, API 20E system and conventional RT-PCR method revealed that the RT-LAMP assay developed in this study is simpler and more rapid than the latter two methods. Therefore, the RT-LAMP method using the easily portable ESE Quant tube scanner can be considered as an effective tool for the rapid screening of V. parahaemolyticus strains in environmental and clinical samples, especially, in remote areas of developing countries during epidemic periods. |
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| Keywords: | Real-time LAMP ESE Quant tube scanner API 20E system Real-time PCR |
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