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

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 diVerent methods of sample preparation. Then samples were analysed by CT specific PCR. The sensitivity of PCR was the basis of ranking of the eight diVerent 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.

Synovial Fluid Changes in Induced Infectious Arthritis in Calves

“…10,25,26 Primary reports in this study did not demonstrate any differences between centrifuged and noncentrifuged samples. These samples originated from only 1 site in the joints, and consequently may not have cellular and bacterial components representative of the entire joint, thus explaining certain negative results.…”

Optimized testing for C. trachomatis DNA in synovial fluid samples in clinical practice

“…DNA in SF [6,9], in order to develop a test procedure applicable in routine diagnostic settings. Altogether, alkaline lysis and the QI-AEX II Gel Extraction Kit® + CTAB reproducibly gave the highest detection rates in the C. tr.…”

“…This procedure used a commercial DNA purification kit with a cetyltrimethylammonium bromide (CTAB) modification supplied by the manufacturer (Qiagen, Hilden, Germany); preparations were performed according to the manufacturer's instructions and as described previously by Kuipers et al [9]: SF pellets were incubated in the supplied digestion buffer (0.1 M NaCl, 1 mM EDTA, 10 mM TRISHCl pH 8, 0.5% Tween 20) containing proteinase K (100 µg/ml) and incubated at 56°C over night [9]. Subsequently, 20 µl 5 mM NaCl was added and samples were mixed thoroughly, followed by addition of 18 µl CTAB solution and incubation for 10 min at 65°C.…”

“…major outer membrane protein gene omp1; all assays were performed using the conditions described by Kuipers et al [9], with the modification that the PCR reaction was reduced to 45 μl and the amount of DNA preparation added was reduced to 5 μl. This is a nested PCR system, with the outer PCR comprising 40 cycles and the inner PCR comprising 25 cycles; the product size is 152 bp.…”

Diagnosing reactive arthritis: Role of clinical setting in the value of serologic and microbiologic assays

“…In the case of a 12% pretest probability, a positive finding on PCR in synovial fluid results in a 73% posttest probability of Chlamydiainduced arthritis but can be as high as 90% if a pretest probability of 30% is assumed. It has to be stressed that there is no agreement at the moment on the optimum technique to detect Chlamydia by PCR (31,32). Furthermore, none of the commercially available tests is sensitive enough to detect chlamydial DNA reliably in synovial fluid when compared with the amplification methods used in these studies, such as nested PCR (33).…”