Abstract:

A rapid one-step method to detect foodborne pathogens such as Salmonella spp., Staphylococcus aureus and
Listeria monocytogenes in foods was developed multiple LAMP (loop-mediated isothermal amplification). According to the
highly conservative gene invA of Salmonella spp., the nuc gene of S. aureus and the hly gene of L. monocytogenes, three sets
of primers were designed and synthesized. Eight representative test strains were used in LAMP development. The optimized
LAMP reaction system was as follows: Mg2+ concentration of 4.6 mmol/L and reaction temperature of 61 ℃. Electrophoresis
of LAMP products, observation of the centrifugal precipitate and fluorescent staining with SYBR Green I indicated that three
pathogens could be detected within 90 min in one step by using this LAMP system. The LAMP assay showed a sensitivity
of 10 fg/μL, which is 1000 times higher than that of conventional PCR detection (10 pg/μL). The specificity was verified
by using 20 strains of non-targeting bacteria including L. innocua as negative controls. The sequence analysis of LAMP
products confirmed that this assay could correctly amplify the target gene in accordance with the design presented in this
study. Overall, the novel multiple LAMP method reported herein is characteristics of rapid detection, high specificity and
excellent sensitivity, which can be applied in the rapid detection of Salmonella spp., S. aureus and L. monocytogenes.

Key words: loop-mediated isothermal amplification (LAMP), Salmonella spp., Staphylococcus aureus, Listeria monocytogenes, rapid detection