LAMP assay to detect Vibrio parahaemolyticus causing acute hepatopancreatic necrosis disease in shrimp

Acute hepatopancreatic necrosis disease (AHPND) is a serious disease in shrimp and results in considerable losses for the shrimp aquaculture industry. The etiologic agent of AHPND has recently been identified as a unique strain of Vibrio parahaemolyticus: One that is different from human pathogenic V. parahaemolyticus strains. In this study, two sets of primers (LAMP-A2 and LAMP-A3) were developed and validated for use in a LAMP assay to specifically identify V. parahaemolyticus causing AHPND (V. parahaemolyticus AHPND). LAMP-A2 and LAMP-A3 detected all 33 V. parahaemolyticus AHPND isolates except the non-V. parahaemolyticus AHPND isolates and 19 other closely related bacterial species. In pure culture and in spiked shrimp experiments, the LAMP assay was superior to PCR for the detection of V. parahaemolyticus AHPND. In pure cultures, the detection limit of LAMP-A3 was 53 CFU/ml or 0.1 CFU per reaction (10x lower than LAMP-A2), whereas in spiked shrimp experiments, the detection limit was 4.4 x 10(5) CFU/ml or 8.8 x 10(2) CFU per reaction. Further testing of 24 post-larvae, shrimp, sediment and water samples collected from a shrimp farm revealed that V. parahaemolyticus AHPND was detected mostly in the sediment samples. Taken together, the results suggested that the LAMP-A3-based LAMP assay, but not LAMP-A2 or PCR, was suitable for the identification of V. parahaemolyticus AHPND in shrimp aquaculture.