A Duplex PCR Assay for Simultaneously Detecting Two Virulent Strains of Staphylococcus aureus in Rabbits

  Objective  A duplex PCR assay for simultaneous detection on hyper- and low-virulent Staphylococcus aureus strains in rabbits was developed.

  Methods  The assay was based on the specific primers targeting the nuc and pvl genes in a hyper-virulent and a low-virulent strain of S. aureus isolated from rabbits in Fujian. Primer concentration and annealing temperature for the assay were optimized, and detection specificity, sensitivity, and accuracy determined.

  Results  The optimized primer concentration was 0.6 μmol·L⁻¹ and annealing temperature 59.6 ℃ for the assay. Only the hyper- and low-virulent S. aureus isolated from rabbits were detected with no cross-reactions on the commonly found pathogens, such as Pasteurella multocida , Bordetella bronchiseptica, Klebsiella pneumonia , and Escherichia coli , nor on the negative control. The methodology was highly sensitive with a detection limit of 10 pg genomic DNA on the hyper-virulent strain and 100 fg on the low-virulent strains. The coefficients of variation of intra- and inter-assay were 0 indicating high repeatability on both applications. In addition, the test results of the duplex PCR assay were 100% coincident with those obtained by the PCR assays targeting nuc and pvl individually.

  Conclusion  The newly developed duplex PCR assay could simultaneously detect the two virulent S. aureus strains with high specificity, sensibility, repeatability, and accuracy.