Development of a novel, rapid processing protocol for polymerase chain reaction-based detection of bacterial infections in synovial fluids.
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 with Escherichia coli and Staphyloccocus aureus. We have applied this new methodology to clinical SF specimens with results superior to standard laboratory culturing assays.
Mariani
B D
BD
Department of Orthopaedic Surgery, Thomas Jefferson University, Philadelphia, PA 19107, USA.
Levine
M J
MJ
Booth
R E
RE
Tuan
R S
RS
D001424:Bacterial Infections / Q000175:diagnosis* / Q000209:etiology / Q000235:genetics; D001483:Base Sequence; D015139:Blotting, Southern; D004269:DNA, Bacterial / Q000302:isolation & purification*; D004587:Electrophoresis, Agar Gel; D004926:Escherichia coli / Q000737:chemistry* / Q000235:genetics; D006801:Humans; D008969:Molecular Sequence Data; D009693:Nucleic Acid Hybridization; D016133:Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D013211:Staphylococcus aureus / Q000737:chemistry / Q000235:genetics; D013582:Synovial Fluid / Q000382:microbiology*
D004269:DNA, Bacterial