title: Development and evaluation of a PCR-based immunoassay for the rapid detection of methicillin-resistant Staphylococcus aureus.
abstract: A multiplex polymerase chain reaction (PCR), involving detection of the mecA and femB genes, was combined with a novel immunoassay system capable of detecting specific PCR products. The resulting PCR-immunoassay was evaluated in comparison with conventional microbiological techniques used in the routine diagnostic laboratory for the rapid identification of methicillin-resistant Staphylococcus aureus (MRSA), either in pure culture or in overnight broth cultures obtained following enrichment of patient screening swabs. Among the 480 purified isolates of staphylococci and 246 enrichment broths examined, only one 'false-negative' result was obtained by PCR, compared with 18 'false-negative' results obtained by conventional methodology and demonstrated by further conventional examination. Five demonstrable 'false-positive' results were obtained by conventional methodology, compared with a possible 10 by the PCR-immunoassay, although it was not certain that these 10 PCR results were true 'false positives' as, by definition, MRSA could not be isolated by conventional methodology. The results indicated that the routine diagnostic laboratory was encountering difficulties in identifying MRSA correctly, and that the conventional microbiological techniques lacked sensitivity. Overall, the PCR technique was more accurate and sensitive than conventional methodology in detecting MRSA, and results were available within 24 h of screening swabs arriving in the laboratory, compared with a minimum of 48-72 h by conventional techniques. The immunoassay system added to the usefulness of the method by allowing the detection of specific PCR products within 5 min of completing the PCR, without the normal additional step of agarose gel electrophoresis.
authors.affiliation: Department of Microbiology and PHLS Laboratory, University Hospital, Queen's Medical Centre, Nottingham. Kevin.Towner@nott.ac.uk
authors.forename: K J
authors.initials: KJ
authors.lastname: Towner
authors.forename: D C
authors.initials: DC
authors.lastname: Talbot
authors.forename: R
authors.initials: R
authors.lastname: Curran
authors.forename: C A
authors.initials: CA
authors.lastname: Webster
authors.forename: H
authors.initials: H
authors.lastname: Humphreys
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics; D002353:Carrier State / Q000175:diagnosis / Q000382:microbiology; D003428:Cross Infection / Q000175:diagnosis / Q000382:microbiology* / Q000517:prevention & control; D004269:DNA, Bacterial / Q000032:analysis / Q000302:isolation & purification; D005069:Evaluation Studies as Topic; D005188:False Negative Reactions; D006801:Humans; D007118:Immunoassay; D017053:Infection Control / Q000379:methods; D008403:Mass Screening / Q000379:methods; D016106:Methicillin Resistance / Q000235:genetics; D016133:Polymerase Chain Reaction; D013203:Staphylococcal Infections / Q000175:diagnosis / Q000382:microbiology* / Q000517:prevention & control; D013211:Staphylococcus aureus / Q000187:drug effects / Q000235:genetics / Q000302:isolation & purification*
chemical_list: D001426:Bacterial Proteins; D004269:DNA, Bacterial; C102928:FemB protein, Staphylococcus epidermidis