AIMS: The current study was aimed to develop a loop-mediated isothermal amplification (LAMP) combined with amplicon detection by chromatographic lateral flow dipstick (LFD) assay for rapid and specific detection of Vibrio parahaemolyticus. METHODS AND RESULTS: Biotinylated LAMP amplicons were produced by a set of four designed primers that recognized specifically the V. parahaemolyticus thermolabile haemolysin (tlh) gene followed by hybridization with an FITC-labelled probe and LFD detection. The optimized time and temperature conditions for the LAMP assay were 90 min at 65 °C. The LAMP-LFD method accurately identified 28 isolates of V. parahaemolyticus but did not detect 24 non-parahaemolyticus Vibrio isolates and 35 non-Vibrio bacterial isolates. The sensitivity of LAMP-LFD for V. parahaemolyticus detection in pure cultures was 120 CFU ml⁻¹. In the case of spiked shrimp samples without enrichment, the detection limit for V. parahaemolyticus was 1·8 x 10³ CFU g⁻¹ or equivalent to 3 CFU per reaction while that of conventional PCR was 30 CFU per reaction. CONCLUSIONS: The established LAMP-LFD assay targeting tlh gene was specific, rapid and sensitive for identification of V. parahaemolyticus. SIGNIFICANCE AND IMPACT OF THE STUDY: The developed LAMP-LFD assay provided a valuable tool for detection of V. parahaemolyticus and can be used effectively for identification of V. parahaemolyticus in contaminated food sample.
AIMS: The current study was aimed to develop a loop-mediated isothermal amplification (LAMP) combined with amplicon detection by chromatographic lateral flow dipstick (LFD) assay for rapid and specific detection of Vibrio parahaemolyticus. METHODS AND RESULTS: Biotinylated LAMP amplicons were produced by a set of four designed primers that recognized specifically the V. parahaemolyticus thermolabile haemolysin (tlh) gene followed by hybridization with an FITC-labelled probe and LFD detection. The optimized time and temperature conditions for the LAMP assay were 90 min at 65 °C. The LAMP-LFD method accurately identified 28 isolates of V. parahaemolyticus but did not detect 24 non-parahaemolyticus Vibrio isolates and 35 non-Vibrio bacterial isolates. The sensitivity of LAMP-LFD for V. parahaemolyticus detection in pure cultures was 120 CFU ml⁻¹. In the case of spiked shrimp samples without enrichment, the detection limit for V. parahaemolyticus was 1·8 x 10³ CFU g⁻¹ or equivalent to 3 CFU per reaction while that of conventional PCR was 30 CFU per reaction. CONCLUSIONS: The established LAMP-LFD assay targeting tlh gene was specific, rapid and sensitive for identification of V. parahaemolyticus. SIGNIFICANCE AND IMPACT OF THE STUDY: The developed LAMP-LFD assay provided a valuable tool for detection of V. parahaemolyticus and can be used effectively for identification of V. parahaemolyticus in contaminated food sample.
Authors: Miriam Jauset-Rubio; Markéta Svobodová; Teresa Mairal; Calum McNeil; Neil Keegan; Ayman Saeed; Mohammad Nooredeen Abbas; Mohammad S El-Shahawi; Abdulaziz S Bashammakh; Abdulrahman O Alyoubi; Ciara K O Sullivan Journal: Sci Rep Date: 2016-11-25 Impact factor: 4.379