Purpose: To evaluate multiplex high-resolution melting analysis assays as a simple method for preliminary identification of Salmonella serovars.
Methods: Salmonella cultures (95 unique strains; 62 unique serovars) were grown in tryptic soy broth (37°C, 24 h) prior to DNA extraction (wash, spin, boil method). Two multiplex HRM assays (HRM-STM, HRM-STY) were developed targeting loci previously used for differentiating Salmonella serovars. Together, the two HRM assays target ten unique sequences present in some combination in many Salmonella spp. The HRM assays were prepared using MeltDoctor HRM Master Mix, 200 nm of each primer, and 2 μl of template DNA. PCR was performed on the ABI7500 Fast using the SDS software (v1.4) with an initial denaturation of 95°C for 10 min followed by 40 cycles of 95°C for 15 s and 60°C for 60 s. Melt curve data was collected using standard dissociation stage settings and analyzed using HRM software (v2.0).
Results: The HRM-STM assay effectively grouped the Salmonella strains into 12 broad groups and easily differentiated Typhimurium, Enteritidis, and Montevideo (among others). Typhimurium and Saintpaul could be differentiated when HRM-STM and HRM-STY were compared in combination. Similarly, Enteritidis could be differentiated from Hadar, Cubana, Mbdanka, and Thompson using both assays. Enteritidis/Dublin and Kentucky/Thompson strains could not be differentiated with the combination assays. Strains representing Montevideo, Tennessee, Senftenberg, and Kentucky serovars showed large variability in HRM results.
Significance: Multiplex high-resolution melting analysis is useful for preliminary identification of Salmonella serovars. The assay(s) may provide a greater service as a screening tool to differentiate unique isolates from environmental or food sources prior to more expensive and time-consuming methods (e.g., serotyping/fingerprinting).