High-resolution melting curve FRET-qPCR rapidly distinguishes Streptococcus equi subsp. equi and zooepidemicus

ABSTRACT We developed a fluorescence resonance energy transfer (FRET)-based real-time PCR (qPCR) assay coupled with high-resolution melting (HRM) analysis to differentiate Streptococcus equi subsp. equi (SEE) from S. equi subsp. zooepidemicus (SEZ), two closely related equine pathogens with distinct clinical implications. The assay targets a region of the 23S rRNA gene containing single-nucleotide polymorphisms that differentiate the subspecies. By combining subspecies-specific probes with melting temperature ( T m ) analysis, the assay accurately distinguished SEE, SEZ, and mixed infections without the need for post-PCR processing. The entire workflow—from DNA extraction to result interpretation—was completed in under 2 h. The assay demonstrated high sensitivity and specificity, detecting as few as one genome copy per reaction, and showed excellent reproducibility with 100% concordance to whole-genome sequencing (WGS) results. Notably, one nasal swab isolate biochemically identified as SEE (based on its fermentation profile) was reclassified as SEZ by the FRET-qPCR assay, a result confirmed by WGS. Among 127 equine clinical samples, SEE was detected in 18% (23/127) and SEZ in 82% (104/127). SEZ predominated in eye, sinus, transtracheal aspirate, uterine, and wound samples, whereas SEE was significantly associated with guttural pouch samples. This FRET-qPCR assay offers a rapid, accurate, and practical molecular diagnostic tool for distinguishing S. equi subspecies, supporting improved clinical management and outbreak control in equine populations. IMPORTANCE Accurately identifying the cause of respiratory infections in horses is essential for proper treatment and preventing outbreaks. This study introduces a rapid and reliable test that distinguishes between Streptococcus equi subsp. equi (which causes the serious disease strangles) and its close relative S. equi subsp. zooepidemicus (which usually causes milder, less contagious infections). Traditional tests can be slow or give unclear results, especially for unusual strains. Our new one-step molecular test uses melting curve analysis to rapidly and accurately differentiate between these bacterial subspecies—even in mixed infections. The method, which matches the accuracy of whole-genome sequencing but is much faster and easier to use, can help veterinarians to make more accurate diagnoses and improve tracking and control of equine respiratory disease.