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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Levine, M., Mariani, B., Tuan, R., and Booth Jr., R. (1995) Molecular genetic diagnosis of infected total joint arthroplasty.J. Arthropl. 10, 93,94.
Mullis, K. and Faloona, F. (1987) Specific synthesis of DNAin vitro via a polymerase-catalyzed chain reaction.Methods Enzymol. 155, 335–350.
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.
Southern, E. (1975) Detection of specific sequences among DNA fragments separated by gel electrophoresis.J. Mol. Biol. 98, 503–517.
Griffin, H. and Griffin, A. (1994) PCR Technology: current Innovations, CRC, Boca Raton, FL.
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.
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.
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.
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.
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.
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Mariani, B.D., Levine, M.J., Booth, R.E. et al. Development of a novel, rapid processing protocol for polymerase chain reaction-based detection of bacterial infections in synovial fluids. Mol Biotechnol 4, 227–237 (1995). https://doi.org/10.1007/BF02779016
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DOI: https://doi.org/10.1007/BF02779016