title: Development and evaluation of hexaplex PCR for rapid detection of methicillin, cadmium/zinc and antiseptic-resistant staphylococci, with simultaneous identification of PVL-positive and -negative Staphylococcus aureus and coagulase negative staphylococci.
abstract: We developed a multiplex PCR to detect the presence of methicillin- (mecA), cadmium/zinc-(czrC) and antiseptic-resistant (qacA/B) staphylococci and to identify Panton-Valentine leukocidin (PVL)-positive and -negative Staphylococcus aureus and coagulase-negative staphylococci (CoNS) from infected and healthy eyes. The assay was validated on 177 staphylococci comprising of 55 each of S. aureus and CoNS isolated from infected eyes and five S. aureus and 62 CoNS isolated from healthy eyes and nine direct ocular samples. Nine direct ocular samples for in situ testing consisted of corneal scrapings (4), conjunctiva swabs (2) and others (3). Multiplex PCR result was correlated with genotype data obtained with single PCR and dot-blot assay. The control strains that were positive in multiplex PCR for 16S rRNA, nuc, mecA, pvl, czrC and qacA/B genes were also positive in the dot-blot assay. The specificity of amplified genes obtained with reference strains was further confirmed by DNA sequencing. The single step-hexaplex PCR method can be used for rapid detection of mecA, nuc, pvl, czrC and qacA/B genes in staphylococci with simultaneous identification of PVL-positive and -negative S. aureus and CoNS from a variety of ocular samples.
authors.affiliation: Infectious Disease Biology, Institute of Life Sciences, Bhubaneswar, Orissa, India.
authors.forename: Sasmita
authors.initials: S
authors.lastname: Panda
authors.forename: Sarita
authors.initials: S
authors.lastname: Kar
authors.forename: Ranginee
authors.initials: R
authors.lastname: Choudhury
authors.forename: Savitri
authors.initials: S
authors.lastname: Sharma
authors.forename: Durg V
authors.initials: DV
authors.lastname: Singh
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism; D001427:Bacterial Toxins / Q000235:genetics* / Q000378:metabolism; D015373:Bacterial Typing Techniques / Q000379:methods; D003030:Coagulase / Q000235:genetics* / Q000378:metabolism; D005098:Exotoxins / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D007956:Leukocidins / Q000235:genetics* / Q000378:metabolism; D026901:Membrane Transport Proteins / Q000235:genetics* / Q000378:metabolism; D046915:Penicillin-Binding Proteins; D016133:Polymerase Chain Reaction / Q000379:methods*; D013203:Staphylococcal Infections / Q000382:microbiology*; D013210:Staphylococcus / Q000145:classification / Q000187:drug effects / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D013211:Staphylococcus aureus / Q000145:classification / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D001427:Bacterial Toxins; D003030:Coagulase; D005098:Exotoxins; D007956:Leukocidins; D026901:Membrane Transport Proteins; C078284:Panton-Valentine leukocidin; D046915:Penicillin-Binding Proteins; C487789:mecA protein, Staphylococcus aureus; C068498:qacA protein, Staphylococcus aureus
keywords: Panton-Valentine leukocidin; Staphylococcus aureus; cadmium/zinc and antiseptic resistance; coagulase negative staphylococci; hexaplex PCR; methicillin