title: Quantitative PCR for detection and discrimination of the bloodborne pathogen Staphylococcus epidermidis in platelet preparations using divIVA and icaA as target genes.
abstract: Bacterial contamination of blood components is the major microbiological cause of transfusion-associated morbidity, with Staphylococcus epidermidis being the most frequently isolated organism from contaminated platelet preparations (PPs). We have recently shown that S. epidermidis forms biofilms during platelet storage, which might account for reported missed detection during routine screening. In this study, we developed a highly sensitive and specific multiplex quantitative PCR (QPCR) assay to detect S. epidermidis in PPs at levels of 10(2)-10(3) cfu/mL. A specific primer pair and hydrolysis probe were designed to amplify an internal region of the cell division divIVA gene that is unique to S. epidermidis. In addition, an internal sequence of the virulence gene icaA, which is involved in the synthesis of the S. epidermidis biofilm matrix, was selected to allow for differentiation of potentially biofilm-forming S. epidermidis isolates. A conserved region of the 8 alleles of the HLA-DQalpha1 locus present in residual white blood cells in PPs was selected as an internal control for the assay. The specificity of this assay was confirmed, as other staphylococcal species that were tested with the optimized parameters were not detected. This QPCR assay could be adaptable for the detection of other bloodborne bacterial pathogens.
authors.affiliation: Canadian Blood Services, 1800 Alta Vista Drive, Ottawa, ON K1G 4J5, Canada.
authors.forename: Cherie Cameron
authors.initials: CC
authors.lastname: Mastronardi
authors.forename: Sandra
authors.initials: S
authors.lastname: Ramírez-Arcos
mesh_terms: D001426:Bacterial Proteins / Q000032:analysis* / Q000235:genetics; D018441:Biofilms / Q000254:growth & development*; D001792:Blood Platelets / Q000382:microbiology*; D017848:Blood-Borne Pathogens; D015815:Cell Adhesion Molecules / Q000032:analysis* / Q000235:genetics; D018797:Cell Cycle Proteins / Q000032:analysis* / Q000235:genetics; D004269:DNA, Bacterial / Q000032:analysis; D006683:HLA-DQ Antigens / Q000235:genetics; D025202:Molecular Diagnostic Techniques; D017713:Platelet Transfusion; D016133:Polymerase Chain Reaction / Q000379:methods*; D013203:Staphylococcal Infections / Q000382:microbiology; D013212:Staphylococcus epidermidis / Q000235:genetics / Q000302:isolation & purification*
chemical_list: D001426:Bacterial Proteins; D015815:Cell Adhesion Molecules; D018797:Cell Cycle Proteins; D004269:DNA, Bacterial; C104636:DivIVA protein, bacteria; D006683:HLA-DQ Antigens