Multiplex loop-mediated isothermal amplification-based lateral flow dipstick for simultaneous detection of 3 food-borne pathogens in powdered infant formula

摘要

In this study, we established a rapid, simple, andsensitive method for visual and point-of-care detectionof SalmoNE_Lla spp., Cronobacter spp., and Staphylococcusaureus in powdered infant formula (PIF) basedon multiplex loop-mediated isothermal amplification(mLAMP) combined with lateral flow dipstick (LFD).Three different species-specific target genes, siiA ofSalmoNE_Lla spp., internal transcribed space (ITS) ofCronobacter spp., and nuc of Staph. aureus, were appliedin the mLAMP with biotin-, digoxin-, and TexasRed-modified forward inner primers and fluorescein isothiocyanate(FITC)-modified backward inner primers.After mLAMP, a large number of modified ampliconswere detected with LFD; one end of the amplicon wasconjugated to the anti-FITC antibody on gold nanoparticlesand the other end to streptavidin (anti-digoxin oranti-Texas Red antibody) on test lines. Visual inspectionof the device relies on the presence of a red bandformed by accumulation of sandwich composites. Thedetection limits of this mLAMP-LFD assay for SalmoNE_Llaspp., Cronobacter spp., and Staph. aureus in PIFwithout enrichment were 4.2, 2.6, and 3.4 cfu/g, respectively.The whole method can be completed in lessthan 1 h. Thus, mLAMP-LFD is a rapid and efficientmethod for simultaneously detecting SalmoNE_Lla spp.,Cronobacter spp., and Staph. aureus in PIF.