Optimised sample preparation of synovial fluid for detection ofChlamydia trachomatis DNA by polymerase chain reaction
- Jens G Kuipersa,
- Lars Nietfelda,
- Ute Dreses-Werringloera,
- Lars Koehlera,
- Juergen Wollenhaupta,
- Henning Zeidlera,
- Michael Hammerb
- aDivision of Rheumatology, Department of Internal Medicine and Dermatology, Hannover Medical School, Hannover, Germany, bDepartment of Rheumatology, Nordwestdeutsches Rheumazentrum, St Josef-Stift, Sendenhorst, Germany
- Dr J Kuipers, Division of Rheumatology, Hannover Medical School, 30623 Hannover, Germany.
- Accepted 13 October 1998
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.
