Rapid and sensitive detection of Vibrio alginolyticus by loop-mediated isothermal amplification combined with a lateral flow dipstick targeted to the rpoX gene

Abstract

Vibrio alginolyticus is a major bacterial pathogen causing disease in marine animals. The present study aimed to develop a loop-mediated isothermal amplification (LAMP) coupled with a lateral flow dipstick (LFD) for rapid and simple visual detection of V. alginolyticus–specific amplicons. The biotin-labeled LAMP amplicons from the targeted portion of a gene encoding rpoS-like sigma factor (rpoX) were generated at 60°C for 1 h and then hybridized with a fluorescein isothiocyanate–labeled probe for 5 min for visual detection with LFD. In pure cultures, the detection limit of the LAMP–LFD technique for V. alginolyticus was 1.8 × 102 CFU/mL while that of PCR was 1.8 × 103 CFU/mL. In spiked whiteleg shrimp samples Penaeus vannamei, the sensitivity for V. alginolyticus detection was 2 × 103 CFU/g (equivalent to 4 CFU per reaction) while PCR was 10 times less sensitive. The LAMP–LFD method for V. alginolyticus correctly identified 21 isolates of V. alginolyticus but did not recognize 23 non-V. alginolyticus Vibrio isolates and 15 non-Vibrio isolates. In summary, this LAMP–LFD method targeted to the rpoX gene is a convenient assay for specific identification of V. alginolyticus with high sensitivity. © American Fisheries Society 2015.