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. 1999 Feb;58(2):103–108. doi: 10.1136/ard.58.2.103

Optimised sample preparation of synovial fluid for detection of Chlamydia trachomatis DNA by polymerase chain reaction

J Kuipers 1, L Nietfeld 1, U Dreses-Werringloe 1, L Koehler 1, J Wollenhaupt 1, H Zeidler 1, M Hammer 1
PMCID: PMC1752829  PMID: 10343525

Abstract

OBJECTIVE—To optimise sample preparation of synovial fluid for Chlamydia trachomatis (CT) specific polymerase chain reaction (PCR).
METHODS—Serial dilutions of purified CT elementary bodies in synovial fluid were prepared. The synovial fluid pellet was processed by eight different methods of sample preparation. Then samples were analysed by CT specific PCR. The sensitivity of PCR was the basis of ranking of the eight different methods.
RESULTS—Highest sensitivity was achieved by methods including an additional step of DNA isolation. Additional extraction of protein and polysaccharides by cetyltrimethylammonium bromide (CTAB) increased sensitivity. Addition of hyaluronidase did not increase sensitivity of QIAEX-DNA extraction but was necessary, however, before phenol-chloroform-DNA extraction.
CONCLUSIONS—The method of synovial fluid sample preparation significantly influences the sensitivity of subsequent PCR. Additional DNA isolation and extraction of PCR inhibitors by CTAB led to higher sensitivity.

 Keywords: Chlamydia trachomatis; polymerase chain reaction; synovial fluid

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Figure 1  .

Figure 1  

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The eight different methods for processing of synovial fluid analysed in this study.

Figure 2  .

Figure 2  

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Number of fluorescent particles (depicted on the y axis) per inclusion forming unit (IFU, shown on the x axis). Dilutions of three randomly selected aliquots from the stock of purified C trachomatis were analysed by immunofluorescence. Three slides per dilution per aliquot were analysed. The calculated curve for linear regression is shown (slope = 7.7, r2 = 0.82, p<0.0001).

Figure 3  .

Figure 3  

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Electrophoresis of amplified DNA by nested PCR for serial 10-fold dilutions of persistently infected PBMo in synovial fluid. Lanes 1 and 9: molecular weight marker (pBR322 cleaved with HaeIII), lane 2: positive control (purified DNA of C trachomatis), lanes 3 and 8: negative controls prepared together with the other samples (lane 3: distilled water, lane 8: synovial fluid from RA patient), lanes 4, 5, 6, and 7: 10-fold serial dilutions of persistently infected PBMo from 102 to 10-1 PBMo per ml of synovial fluid. Left panel: dilution series processed by method (3b). Right panel: dilution series processed by method (4b).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

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