PCR-based detection of Erysipelothrix rhusiopathiae using EvaGreen real-time PCR assay
DOI:
https://doi.org/10.31073/onehealthjournal2026-III-02Keywords:
Erysipelothrix rhusiopathiae, swine erysipelas, EvaGreen, real-time PCR, molecular diagnostics, spaA geneAbstract
The present study aimed to develop and evaluate an EvaGreen real-time PCR assay for rapid, sensitive, and specific detection of Erysipelothrix rhusiopathiae, the causative agent of swine erysipelas and an important zoonotic pathogen.
Species-specific primers targeting the spaA gene of E. rhusiopathiae were used for amplification on the iTower real-time PCR platform (Jena Analytik, Germany). Reaction conditions were optimized using EvaGreen fluorescent chemistry. Analytical sensitivity was determined using tenfold serial dilutions of purified genomic DNA, while specificity was evaluated against non-target bacterial species commonly associated with swine infections. Clinical samples obtained from pigs with suspected erysipelas, including tonsils, spleen, lymph nodes, heart valves, blood, and synovial fluid, were tested to assess diagnostic applicability.
The developed assay successfully detected E. rhusiopathiae DNA in all positive controls and field-positive samples. The limit of detection was established at 10 fg/µL. Standard curve analysis demonstrated excellent linearity (R² = 0.998) and high amplification efficiency (98.4%). No amplification was observed with non-target bacterial DNA, confirming high analytical specificity. All positive samples produced a single characteristic melting peak at 81.7 ± 0.3°C, indicating specific amplification. Among 48 field specimens, 19 samples (39.6%) tested positive, with the highest detection rates observed in tonsillar and splenic tissues. Intra- and inter-assay variation remained below 3%, confirming strong repeatability and reproducibility.
The EvaGreen real-time PCR assay is a rapid, reliable, and cost-effective molecular method for detection of E. rhusiopathiae. Its high sensitivity, specificity, and robust performance make it suitable for routine veterinary diagnostics, outbreak investigations, and epidemiological surveillance of swine erysipelas. The assay may serve as an effective alternative to probe-based real-time PCR systems, particularly in laboratories with limited resources.
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