Loop-Mediated Isothermal Amplification Assay for Visual Detection of Salmonella enterica Serovar Typhimurium in Food Animal Meat Products

Abstract

Detection of Salmonella, a highly diverse foodborne pathogen, is paramount to ensure safety and protection of the animal industry and its consumers. Salmonella enterica serovar Typhimurium is among the most important non-typhoidal serovars causing gastroenteritis worldwide. However, traditional serovar identification is labor- and resource-intensive, while typical molecular tools require expensive reagents and equipment. Hence, this study developed and optimized a calcein-based and closed-tube loop-mediated isothermal amplification (LAMP)-based assay to detect S. Typhimurium following enrichment steps compared with an optimized PCR assay. The PCR assay showed 100% specificity in silico confirmed through DNA sequencing. For actual specificity testing, both PCR and LAMP showed 100% specificity to S. Typhimurium. For DNA sensitivity, while PCR showed a limit of detection of 22 pg/μL, LAMP showed a 100-fold higher sensitivity at 220 fg/μL. Meanwhile, for pure culture sensitivity, both assays detected at least 4.98 × 104 CFU/mL. Parallel testing of 208 raw meat samples from wet markets in Metro Manila, Philippines, showed corroboration and statistical association of the optimized PCR and LAMP with 89.42% and 90.87% positivity rates for S. Typhimurium, respectively. Hence, the developed closed-tube and calcein-based LAMP assay is potentially a powerful yet simple, sensitive, and fast method for S. Typhimurium detection.