Sensitive Molecular Detection for Salmonella Enterica Serovars Typhimurium

Abstract

Introduction: Salmonella infections contribute significantly to gastroenteritis cases, with the National Salmonella Reference Laboratory reporting 500 isolates in 2022. However, traditional culture-based methods for detecting Salmonella in samples can take 4 to 7 days to confirm a positive result, which poses health risks due to delayed detection. Given these health risks, swift and accurate detection methods are essential to minimize both false-positive and false-negative outcomes. Salmonella, a gram-negative bacterium within the Enterobacteriaceae family, demonstrates remarkable hardiness, surviving for several weeks in dry environments and months in water. Although most serotypes of Salmonella cause relatively mild gastroenteritis, some, particularly those transmitted from animals to humans, can lead to severe, life-threatening conditions
 Methods: The qRT-PCR procedure involved the design of primers and probes targeting the same genes as the mPCR assay. These primer sets were reconfigured to generate smaller amplicons suitable for qRT-PCR systems
 Results: qRT-PCR process, TaqMan probes were meticulously designed for specific target genes: FAM dye was employed to detect STM2745, Cy5 dye was used for STM4492, and Rox dye was utilized to detect. A standard curve was constructed using Typhimurium LT2 genomic DNA. Each sample underwent duplicate analysis, and Rotor-Gene software was employed to assign threshold values for each channel.
 Conclusion: The effectiveness of our qPCR assay for the detection of Salmonella across a diverse array of matrices. Notably, our results unveiled distinct limits of detection for Salmonella in various samples. Specifically, a parallel vein, the deployment of a PCR assay, leveraging an immunomagnetic separation technique for DNA extraction, was studied by another group. While subsequent analysis of Salmonella detected via our assay may necessitate the full ISO SMT method for live culture isolation, this supplementary step can be seamlessly conducted alongside qRT-PCR.