FRET-based real-time PCR with high-resolution melting for rapid differentiation of Streptococcus equi subspecies

Background

Molecular diagnostics for equine streptococcal infections rely on nucleic acid amplification techniques to provide rapid, accurate identification of pathogens. Streptococcus equi subsp. equi (SEE) causes strangles, a highly contagious respiratory disease, whereas S. equi subsp. zooepidemicus (SEZ) acts as an opportunistic pathogen in various tissues. Rapid discrimination between these subspecies is critical for outbreak control, epidemiological surveillance, and appropriate therapeutic intervention. Conventional culture-based methods require days and lack subspecies specificity, while standard PCR assays can detect genus-level targets but often fail to resolve single nucleotide differences reliably. A faster, more precise diagnostic is therefore needed to guide clinical and biosecurity decisions.

Current diagnostic workflows depend on culture isolation and biochemical tests that can take 48–72 hours and exhibit variable sensitivity. Molecular assays, including conventional PCR followed by gel electrophoresis or probe-based real-time PCR, typically target conserved regions and lack the resolution to discriminate single nucleotide polymorphisms distinguishing SEE from SEZ. Post-PCR processing steps such as sequencing or restriction fragment analysis introduce delays, increase labor and risk of contamination, and limit throughput. Furthermore, many require high DNA input, reducing detection of low-copy targets in clinical samples. These limitations hinder timely, confident identification needed for effective disease management.

Technology

A novel FRET-based real-time PCR assay targets a single T→C SNP in the 23S rRNA gene of Streptococcus equi to distinguish the two subspecies. Subspecies-specific probes labeled with 6-FAM and LC Red 640 flank the polymorphism, and thermal cycling on a LightCycler 480 II combines 18 high-stringency step-down cycles with 30 relaxed-stringency fluorescence acquisition cycles. High-resolution melting analysis from 45 °C to 80 °C yields distinct melting temperatures—approximately 69 °C for S. equi subsp. equi, 67 °C for S. equi subsp. zooepidemicus and 68 °C for mixed templates—directly in the closed tube. The workflow, from DNA extraction to result, completes in under two hours with single-copy sensitivity, high specificity against non-target streptococci, intra- and inter-assay coefficients of variation below 1.5%, and 100% concordance with whole-genome sequencing across clinical isolates.

This approach is differentiated by its integration of fluorescence resonance energy transfer with high-resolution melting in a single, rapid assay, eliminating post-PCR processing and reducing contamination risk. Its subspecies-specific probes and precise melting-temperature readout enable real-time discrimination of pure and mixed infections in a high-throughput format. Compared with conventional culture, biochemical tests or sequencing, this method delivers faster turnaround, higher sensitivity and reproducibility, and seamless detection of low-abundance targets, facilitating timely and accurate diagnosis in clinical and epidemiological settings.

Benefits

• Rapid turnaround time (results in under 2 hours)
• Ultra-sensitive detection (single-copy sensitivity)
• High specificity and accuracy (100% concordance with whole-genome sequencing)
• No post-PCR processing required
• Excellent reproducibility (intra/inter-assay CV < 1.5%)
• Ability to distinguish mixed SEE/SEZ infections

Applications

• Equine diagnostic test kits
• Veterinary PCR diagnostic services
• Rapid pathogen screening assays
• FRET-PCR reagent kits
• High-resolution melting kits

Additional info

A FRET-based real-time PCR assay targets a T→C SNP in the 23S rRNA gene to differentiate S. equi subsp. equi from S. equi subsp. zooepidemicus. Subspecies-specific 6-FAM donor and LC Red 640 acceptor probes flank the SNP. After 18 high-stringency step-down and 30 relaxed-stringency cycles on a LightCycler 480 II, high-resolution melting from 45 °C to 80 °C yields distinct Tm peaks (~69 °C, ~67 °C, ~68 °C). Single-copy sensitivity, <2 h runtime, 100% concordance with whole-genome sequencing, CV <1.5%.