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%.