Development of real-time Loop-mediated isothermal amplification assay for rapid detection of Salmonella enterica serovars

Abstract

Typhoid fever remains a major global health burden, particularly in regions with inadequate sanitation and safe water supplies. The disease, caused by Salmonella enterica serovars transmitted via the fecal-oral route, can lead to severe systemic infections with life-threatening complications and contribute to chronic carrier states that perpetuate its spread. Traditional diagnostic methods such as blood culture and biochemical assays, though considered gold standards, are labor-intensive and time-consuming, thereby limiting their practical utility in resource-constrained settings. In response, we developed a real-time loop-mediated isothermal amplification (LAMP) assay for the rapid detection of S. enterica using conserved genomic regions as targets. Unlike conventional PCR, which requires expensive thermal cyclers, our LAMP protocol utilizes a water bath to maintain a constant reaction temperature of 63°C for 90 minutes, significantly reducing equipment costs. Amplified products were rapidly detected by the addition of SYBR Green dye, which produced a distinct green fluorescence under blue light, enabling easy visual interpretation. Comparative analysis revealed that the LAMP assay demonstrated superior sensitivity—detecting Salmonella in 90% of isolates—compared to 85% for PCR, with both methods showing high specificity and no cross-reactivity with non-Salmonella species. These results indicate that the LAMP assay offers a rapid, cost-effective, and sensitive alternative for typhoid diagnosis, holding promise for improved disease surveillance and management in low-resource settings.