Dual-Emission Fluorescence Resonance Energy Transfer (FRET) PCR Discriminates Salmonella Pullorum and Gallinarum

<i>Salmonella</i> Pullorum (<i>S.</i> Pullorum) and <i>Salmonella</i> Gallinarum (<i>S.</i> Gallinarum) are two biovars of <i>Salmonella enterica</i> serovar Gallinarum, responsible for pullorum disease and fowl typhoid, which are the most prevalent and pathogenic forms of salmonellosis in poultry in developing countries. Traditional differentiation methods for <i>S</i>. Pullorum and <i>S</i>. Gallinarum are based on distinct clinical manifestations and biochemical traits, given their indistinguishable nature via serological assays alone. Molecular differentiation methods such as allele-specific PCR and dual PCR combined with gel electrophoresis or enzyme digestion have also been used to discriminate <i>S</i>. Pullorum and <i>S</i>. Gallinarum, but the detection efficiency is not high. This investigation introduces a Fluorescence Resonance Energy Transfer (FRET) PCR assay targeting the <i>pegB</i> gene, exclusively found in specific <i>Salmonella</i> serovars such as <i>S</i>. Pullorum and <i>S</i>. Gallinarum, and exhibiting conserved single-nucleotide polymorphisms across these two biovars. High-resolution melting curve analysis demonstrates distinct dissolution profiles, facilitating the precise discrimination of <i>S</i>. Pullorum and <i>S</i>. Gallinarum. This FRET-PCR assay exhibits a detection limit of 10 copies per reaction and has been rigorously validated utilizing 17 reference strains and 39 clinical isolates. The innovation presented herein provides a valuable tool for the rapid differentiation of <i>S</i>. Pullorum and <i>S</i>. Gallinarum, thereby enhancing diagnostic efficiency and molecular surveillance of poultry <i>Salmonella</i>. The developed <i>pegB</i>-targeting FRET-PCR assay presents a promising alternative to current cumbersome and time-consuming diagnostic modalities, offering significant potential for expedited identification and control of <i>Salmonella</i> in poultry and mitigating economic losses associated with <i>Salmonella</i> contamination in poultry production.