Multiple displacement amplification as an adjunct to PCR-based detection of Staphylococcus aureus in synovial fluid
1 Center for Genomic Sciences, Allegheny-Singer Research Institute, Allegheny General Hospital, Pittsburgh, PA, USA
2 Division of Oral/Maxillofacial Surgery, Allegheny General Hospital, Pittsburgh, PA, USA
3 Division of Otolaryngology/Head and Neck Surgery, Allegheny General Hospital, Pittsburgh, PA, USA
4 Division of Plastic Surgery, Allegheny General Hospital, Pittsburgh, PA, USA
5 Department of Microbiology and Immunology, Drexel University College of Medicine, Allegheny Campus, Pittsburgh, PA, USA
6 National Center for Advanced Tribology at Southampton (nCATS), University of Southampton, Highfield, Southampton, SO17 1BJ, UK
BMC Research Notes 2010, 3:259 doi:10.1186/1756-0500-3-259Published: 13 October 2010
Detection of bacterial nucleic acids in synovial fluid following total joint arthroplasty with suspected infection can be difficult; among other technical challenges, inhibitors in the specimens require extensive sample preparation and can diminish assay sensitivity even using polymerase chain reaction (PCR)-based methods. To address this problem a simple protocol for prior use of multiple displacement amplification (MDA) as an adjunct to PCR was established and tested on both purified S. aureus DNA as well as on clinical samples known to contain S. aureus nucleic acids.
A single round of MDA on purified nucleic acids resulted in a > 300 thousand-fold increase in template DNA on subsequent quantitative PCR (qPCR) analysis. MDA use on clinical samples resulted in at least a 100-fold increase in sensitivity on subsequent qPCR and required no sample preparation other than a simple alkali/heat lysis step. Mixed samples of S. aureus DNA with a 103 - 104-fold excess of human genomic DNA still allowed for MDA amplification of the minor bacterial component to the threshold of detectability.
MDA is a promising technique that may serve to significantly enhance the sensitivity of molecular assays in cases of suspected joint infection while simultaneously reducing the specimen handling required.