Multiple displacement amplification as an adjunct to PCR-based detection of <it>Staphylococcus aureus </it>in synovial fluid
<p>Abstract</p> <p>Background</p> <p>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 a...
| Main Authors: | , , , , , , |
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| Format: | Article |
| Language: | English |
| Published: |
BMC
2010-10-01
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| Series: | BMC Research Notes |
| Online Access: | http://www.biomedcentral.com/1756-0500/3/259 |
| Summary: | <p>Abstract</p> <p>Background</p> <p>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 <it>S. aureus </it>DNA as well as on clinical samples known to contain <it>S. aureus </it>nucleic acids.</p> <p>Findings</p> <p>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 <it>S. aureus </it>DNA with a 10<sup>3 </sup>- 10<sup>4</sup>-fold excess of human genomic DNA still allowed for MDA amplification of the minor bacterial component to the threshold of detectability.</p> <p>Conclusion</p> <p>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.</p> |
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| ISSN: | 1756-0500 |