Application of an improved loop-mediated isothermal amplification detection of Vibrio parahaemolyticus from various seafood samples

Abstract

The specificity and sensitivity of an improved loop-mediated isothermal amplification (LAMP) method for rapid detection of Vibrio parahaemolyticus strains from various seafood samples had been developed and evaluated in this study. Six primers, including outer primers and inner primers, were specially designed for recognizing six distinct sequences on the target gene of tlh. The optimal reaction condition was found to be 65°C for 45 min, with the detection limit as 10 CFU/ml. Application of LAMP assays was performed on 416 food borne V. parahaemolyticus strains isolated from various seafood samples, and the total detection rate for LAMP and polymerase chain reaction (PCR) assay was found to be 96.2% (400/416) and 85.6% (356/416). This is the first report of an improved and simple LAMP detection assay on V. parahaemolyticus employed procedures of simple template deoxyribonucleic acid (DNA) preparation, equipment for LAMP reaction (water bath) and direct result determination via observation of color change. In addition, this is also the first application of LAMP detection on this considerable amount of V. parahaemolyticus isolates (416 strains for application together with 105 reference strains for establishment) with the total identification rate as 96.2%, as well as its extensive application to marine fish, shrimp, oyster, mussel, jellyfish, cuttlefish and seaweed samples, with detection rate ranging from 89.5 to 100%. Key words: Loop-mediated isothermal amplification (LAMP), Vibrio parahaemolyticus, seafood samples.