Development of a novel,rapid processing protocol for polymerase chain reaction-based detection of bacterial infections in synovial fluids |
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Authors: | Brian D Mariani Marc J Levine Robert E Booth Rocky S Tuan |
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Institution: | (1) Orthopaedic Research Laboratory, Department of Orthopaedic Surgery, Thomas Jefferson University, 1015 Walnut Street, 19107 Philadelphia, PA |
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Abstract: | We describe the development of a molecular detection system designed for use with synovial fluid (SF)-based infections. The
methodology employs a lysis/extraction procedure that effectively disrupts microorganisms allowing for release of the microbial
DNA and its amplification by polymerase chain reaction (PCR). We tested the effectiveness of adding a mixed-bed, ion-exchange
resin to the extract to remove PCR inhibitory components present in the SF. After centrifugation to separate the resin, DNA
contained in the supernatant is subjected to PCR using oligonucleotide primers designed for broad-spectrum microorganism detection.
Amplification products are analyzed by agarose gel electrophoresis and/or DNA hybridization methodology. We report here the
detection sensitivity and specificity of the protocol using SF inoculated withEscherichia coli andStaphyloccocus aureus. We have applied this new methodology to clinical SF specimens with results superior to standard laboratory culturing assays. |
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Keywords: | Synovial fluid Bacterial infection Polymerase chain reaction Joint arthroplasty Clinical diagnostics |
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