Improvement and evaluation of loop-mediated isothermal amplification combined with chromatographic flow dipstick assays for Vibrio parahaemolyticus

Abstract

Vibrio parahaemolyticus, a major food-borne pathogen, is a gram-negative rod-shaped halophilic bacterium which inhabits marine environments throughout the world. It can pose a threat to humans after the consumption of raw or undercooked seafood. Fast detection is crucial for hindering and controlling V. parahaemolyticus infection. Compared with traditional methods, loop-mediated isothermal amplification (LAMP) is a simple, rapid and versatile method. It can be performed at one temperature without the need for cycling. As a new method in recent years, LAMP combined with a chromatographic flow dipstick (LFD) meets the needs of point-of-care testing without the need for special instruments. It avoids the limitations of LAMP, reduces detection time and increases detection accuracy. Our previous studies have suggested that the optimized LFD method can improve the sensitivity of LAMP detection and shorten the isothermal amplification time during the detection process. In the present study, two LAMP assays were improved to LFD methods, and a LFD targeting 16S23S rRNA gene internal transcribed spacer (ITS) of V. parahaemolyticus was developed. The lower limit for tlh, toxR, ITS LFD assays were detected as 3.1 × 10⁰, 3.1 × 10¹, and 3.1 × 10⁰ CFU respectively, whether in pure cultures or artificially contaminated food samples. The shortest amplification times at the limit of each assay were determined as 20 min, 35 min and 25 min. A heating block was used to perform two (tlh and ITS) LFD assays to detect 20 food samples. Compared to a standard method (GB 4789.7-2013 National Food Safety Standards, Food Microbiology Inspection, Vibrio parahaemolyticus test), tlh and ITS LFD assays showed more MPN (most probable number) results than that of culture. It demonstrated that the improved LFD technology can provide a simple and rapid detection method with high sensitivity and specificity for detection of V. parahaemolyticus.

Keywords: 16S–23S rRNA gene internal transcribed spacer; LAMP combined with a chromatographic flow dipstick; Tlh; Vibrio parahaemolyticus; toxR.