Rapid Detection and Differentiation of Staphylococcus hyicus and Non-hyicus Staphylococci Using a Novel Duplex Real-Time Polymerase Chain Reaction

Exudative epidermitis (EE), also known as greasy pig disease, is primarily caused by toxigenic Staphylococcus hyicus, but non-hyicus staphylococci such as S. chromogenes and S. sciuri have been implicated in EE outbreaks. Although several polymerase chain reaction (PCR) or real-time PCR (qPCR) methods have been developed to detect S. hyicus and some staphylococcal species, no duplex qPCR (dqPCR) assay is currently available to detect and distinguish S. hyicus from non-hyicus staphylococcal species simultaneously. Therefore, this study developed a novel dqPCR assay for the rapid, differential detection of S. hyicus and non-hyicus staphylococci in a single reaction. The developed dqPCR assay detected staphylococcal isolates and differentiated S. hyicus from non-hyicus staphylococci. The limit of detection (LOD) was 5 × 102 CFU/mL for both targets, which was 10-fold lower than the LOD of the reference qPCR and cPCR assays under standardized condition. In a clinical evaluation of 104 bacterial isolates from clinical EE cases, the dqPCR accurately detected all 57 staphylococcal isolates and specifically distinguished 14 S. hyicus isolates from non-hyicus staphylococcal isolates achieving 100% agreement with the matrix-assisted laser desorption/ionization time-of-flight mass spectrometry results. Considering its short turnaround time, specificity, and sensitivity, the dqPCR assay newly developed in this study will be useful for screening and differentiation of S. hyicus and non-hyicus staphylococcal species in swine clinics.