Development of Duplex Loop-Mediated Isothermal Amplification (dLAMP) Combined with Lateral Flow Dipstick (LFD) for the Rapid and Specific Detection of Vibrio vulnificus and V. parahaemolyticus

Abstract

Loop-mediated isothermal amplification (LAMP) is a rapid, specific, and effective DNA amplification assay. In this study, a duplex LAMP (dLAMP) assay combined with chromatographic lateral flow dipstick (LFD) was developed for the simultaneous identification of Vibrio vulnificus and V. parahaemolyticus. Each primer set that comprised F3, B3, FIP, and BIP recognized the V. vulnificus rpoS gene and the V. parahaemolyticus tlh gene. Digoxigenilated and biotinylated LAMP amplicons of V. vulnificus and V. parahaemolyticus, respectively, were hybridized with corresponded fluorescein isothiocyanate (FITC)-labeled probes for LFD detection. The optimum conditions were 90 min at 63°C. The dLAMP–LFD had high specificity to V. vulnificus and V. parahaemolyticus and showed no cross-amplification with 59 isolates of other bacteria. The detection limit of dLAMP–LFD with a pure culture of V. vulnificus was 2.2 × 10⁴ CFU/mL, corresponding to 41 CFU per reaction, which was equal to that of duplex PCR (dPCR). In the case of V. parahaemolyticus, the detection limit of dLAMP–LFD with a pure culture was 2.2 × 10³ CFU/mL, corresponding to 4.1 CFU per reaction, 100 times more sensitive than that of dPCR. In the spiked shrimp samples, the detection limit of dLAMP–LFD for V. vulnificus was 2.2 × 10⁵ CFU/g, corresponding to 410 CFU per reaction, 10 times more sensitive than that of dPCR. In the case of V. parahaemolyticus, the detection limit of dLAMP–LFD in spiked shrimp samples was 2.2 × 10⁴ CFU/g, corresponding to 41 CFU per reaction, 100 times more sensitive than that of dPCR. These results demonstrate that the simple dLAMP–LFD assay had high specificity and high sensitivity for the concurrent detection of V. vulnificus and V. parahaemolyticus.

Received December 8, 2015; accepted March 28, 2016

Acknowledgments

This work was supported by the National Research Council of Thailand to PC and by the Science Achievement Scholarship of Thailand to PP. The authors thank all of the institutes listed in Table 1 for kindly supplying various species of bacteria.