Real time multiplex PCR assay for the detection of Staphylococcus aureus using nuc and eap genes

Open Access
Luley, Erin Hope
Area of Honors:
Veterinary and Biomedical Sciences
Bachelor of Science
Document Type:
Thesis Supervisors:
  • Bhushan M Jayarao, Thesis Supervisor
  • Lester C Griel Jr., Honors Advisor
  • Real time multiplex PCR
  • Mastitis
  • Staphylococcus aureus
Mastitis is a major economic concern for the dairy industry. Costs incurred from mastitis infections come from reduced production, increased culling rates, and the cost of treatment. Staphylococcus aureus is a gram positive bacterium that is a common cause of mastitis in dairy cattle. S. aureus is part of normal skin flora, but it can migrate into the mammary tissue during milking causing infection and inflammation. A high throughput diagnostic assay with high sensitivity and specificity is needed for timely and accurate diagnosis of infection. A multiplex real time PCR assay was developed for the detection of S. aureus thermonuclease (nuc) and extracellular adherence protein (eap) encoding genes. These two genes are highly prevalent in all S. aureus strains, but not in other Staphylococcus species. The assay was standardized with American Type Culture Collection reference strains and field strains provided by the Pennsylvania Animal Diagnostic Laboratory (University Park, PA). Of these 40 field strains, 38 were positive for eap and 36 positive for nuc based on the PCR assay. Sensitivity of the assay was found to be 95.8% for eap and 89.6% for nuc. Specificity was 100% for both genes. The assay was validated with quarter milk samples from mastitis cases provided by the Pennsylvania Animal Diagnostic Laboratory (University Park, PA). However, the assay was unsuccessful at detecting S. aureus in DNA extracted from milk samples. Once standardized and validated, PCR is a faster, more accurate diagnostic tool than traditional culture methods.