Versatility of a Salmonella Loop-Mediated Isothermal Amplification Assay Using Multiple Platforms and Master Mixes in Animal Food Matrices

Improvement in Salmonella detection methods greatly enhances the efficiency of various food testing programs. A Salmonella loop-mediated isothermal amplification (LAMP) assay has been validated in animal food through multi-laboratory validation. The study aimed to demonstrate the versatility of this molecular assay while expanding it to multiple platforms and various reagent choices for use in animal food testing. Following the U.S. Food and Drug Administration (FDA)'s Methods Validation Guidelines for Microbial Pathogens, we examined the inclusivity, exclusivity, and limit of detection (LOD) of the assay using two platforms (7500 Fast and Genie II) and three LAMP master mixes (GspSSD, GspSSD2.0, and WarmStart) in seven animal food matrices (dry cat food, dry dog food, cattle feed, dairy feed, horse feed, poultry feed, and swine feed). The FDA's Bacteriological Analytical Manual (BAM) Salmonella culture method was the reference method. Inclusivity and exclusivity data were consistent among all six platform and master mix combinations with a few exceptions. Comparable LODs were observed down to the single cell level (WarmStart was 10-fold less sensitive). Performance was similar to the BAM method for detecting fractional positive results in seven animal food matrices. Nonetheless, LAMP time to positive results and annealing/melting temperature differed among master mixes and platforms. The Salmonella LAMP assay was successfully validated in two platforms and three master mixes, making it a flexible tool for use by the FDA's field laboratories in regulatory testing of animal food and for adoption by other food testing programs. We demonstrated the LAMP assay's versatility on two platforms and three master mixes for the rapid and reliable screening of Salmonella in seven animal food matrices. GspSSD2.0 was the fastest master mix (time to positive results as early as 3.5 min) while Genie II had several attractive features from a user perspective.