Visual detection of Shigella based on loop-mediated isothermal amplification combined with a lateral flow dipstick

Abstract:

[Objective] To develop an LAMP-LFD method to detect rapidly Shigella spp., based on nucleotide enrichment by a loop-mediated isothermal amplification (LAMP) and chromatographic visualization by a lateral flow dipstick assay. [Methods] Three pairs of primers were designed based on conserved regions of the invasive plasmid antigen H (ipaH) gene of Shigella flexneri and used in LAMP reaction, among which the forward inner primer Sfl-ipaH-FIP was biotinylated. Similarly, a fluorescein isothiocyanate (FITC)-labeled probe Sfl-ipaH-HP was designed to specifically hybridize with LAMP products. And the hybridized products were visually detected by LFD. [Results] The LAMP-LFD method to detect Shigella spp. was developed. The optimized condition for LAMP reaction was at 63 °C for 40 min; and plus visually detecting by LFD, it was approximately 50 min. The LAMP-LFD method discriminated S. flexneri from another 4 pathogenic bacteria causing diarrhea (Salmonella enteric, Campylobacter jejuni, Yersinia enterocolitica, and Vibrio cholera) and 5 common food-borne pathogens (V. parahaemolyticus, V. vulnificus, Listeria monocytogenes, Staphylococcus aureus, and V. alginolyticus), and 4 different Escherichia coli strains. The detection limit of LAMP-LFD was 1.0×102 CFU/mL for Shigella pure cultures (equivalent to 4 CFU per reaction), which was 100 times lower than that of the conventional PCR method using primers Sfl-ipaH-F3/Sfl-ipaH-B3. In the case of artificially contaminated Common carp intestinal tissue, the detection limit was 5×102 CFU/mL (equivalent to 20 CFU per reaction). [Conclusion] This rapid and accurate LAMP-LFD method is a promising alternative in the routine surveillance and point-of-care test of Shigella spp.