Development of a novel, rapid processing protocol for polymerase chain reaction-based detection of bacterial infections in synovial fluids
- 77 Downloads
- 36 Citations
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.
Index Entries
Synovial fluid Bacterial infection Polymerase chain reaction Joint arthroplasty Clinical diagnosticsPreview
Unable to display preview. Download preview PDF.
References
- 1.Levine, M., Mariani, B., Tuan, R., and Booth Jr., R. (1995) Molecular genetic diagnosis of infected total joint arthroplasty.J. Arthropl. 10, 93,94.CrossRefGoogle Scholar
- 2.Mullis, K. and Faloona, F. (1987) Specific synthesis of DNAin vitro via a polymerase-catalyzed chain reaction.Methods Enzymol. 155, 335–350.PubMedCrossRefGoogle Scholar
- 3.Avaniss-Aghanjani, E., Jones, K., Chapman, D., and Brunk, C. (1994) A molecular technique for identification of bacteria using small subunit ribosomal RNA sequences.Bio Techniques 17, 144–149.Google Scholar
- 4.Southern, E. (1975) Detection of specific sequences among DNA fragments separated by gel electrophoresis.J. Mol. Biol. 98, 503–517.PubMedCrossRefGoogle Scholar
- 5.Griffin, H. and Griffin, A. (1994) PCR Technology: current Innovations, CRC, Boca Raton, FL.Google Scholar
- 6.de Lamballerie, X., Zandotti, C., Vignoli, C., Bollet, C., and de Micco, P. (1992) A one-step microbial DNA extraction method using “Chelex 100” suitable for gene amplification.Res. Microbiol. 143, 785–790.PubMedCrossRefGoogle Scholar
- 7.Muralidhar, B, Rumore, P., and Steinman, C. (1994) Use of the polymerase chain reaction to study arthritis due toNeisseria gonorrhoeae.Arthr. Rheum. 37, 710–717.CrossRefGoogle Scholar
- 8.Brosius, J., Palmer, M., Kennedy, P., and Noller, H. (1978) Complete nucleotide sequence of a 16S ribosomal RNA gene fromEscherichia coli.Proc. Natl. Acad. Sci. USA 75, 4801–4805.PubMedCrossRefGoogle Scholar
- 9.Greisen, K., Loefelholz, M., Purohit, A., and Leong, D. (1994) PCR primers and probes for the 16S rRNA gene of most species of pathogenic bacteria, including bacteria found in cerebrospinal fluid.J. Clin. Microbiol. 32, 335–351.PubMedGoogle Scholar
- 10.Ausubel, F., Brent, R., Kingston, R., Moore, D., Seidman, J., Smith, J., and Struhl, K. (1989)Current Protocols in Molecular Biology, Wiley, New York, pp. 2.9.15–2.9.20.Google Scholar