Abstract
IntroductionPolymerase chain reaction (PCR) and ligase chain reaction (LCR) are used in research for detection of Chlamydia trachomatis (C. tr.) in synovial fluid (SF). However there is no standardized system for diagnostic use in clinical practice, therefore this study aimed at determining the molecular biology method best suited to detect C. tr. from SF.MethodsSF samples were spiked with C. tr. elementary bodies (EB) and human peripheral blood monocytes (PBMo) persistently infected with C. tr. in vitro to evaluate the sensitivity of different molecular biology methods and assays. Five different DNA-extraction methods were tested: 1) Alkaline lysis, 2) QIAex II Gel Extraction Kit®+ CTAB, 3) Chelex®-extraction, 4) QIAmp Tissue Kit® and 5) QIAmp DNA Stool Kit®. All DNA extracts were subjected to 5 different DNA amplification systems to detect C. tr.- DNA in the spiked SF samples: two C. tr. -omp1-- directed PCR, one C. tr.-plasmid-PCR, one C. tr. -16s RNA directed PCR, and one commercially available LCR (LCX®, Abbott laboratories).ResultsIn SF samples spiked with C. tr.-EB and with C. tr.-PBMo, alkaline lysis, detecting 1 C. tr.-EB/ml SF, 0,1 C. tr.-PBMo/ml SF and QIAmp gel extraction kit®+ CTAB detecting 0,1 C. tr. -EB/ml SF, 1 C. tr.-PBMo/ml, respectively, allowed most sensitive detection of the organism in combination with the C. tr.- omp1-(152 bp) PCR. Sensitivity decreased in all methods after storage of the DNA of C. tr.- dilution series at -20°C for 4 months by at least one log phase.ConclusionsThe sensitivity to detect C. tr.- DNA from SF is highly dependent on the DNA extraction method and the detection system applied. Alkaline lysis as well as the QIAmp Gel extraction kit® + CTAB in combination with C. tr.- omp1 - (152 bp) PCR evolved as the most sensitive methods to identify C. tr. in serial dilutions.
Highlights
Polymerase chain reaction (PCR) and ligase chain reaction (LCR) are used in research for detection of Chlamydia trachomatis (C. tr.) in synovial fluid (SF)
In SF samples spiked with C. tr.-elementary bodies (EB) and with C. tr.PBMo, alkaline lysis, detecting 1 C. tr.-EB/ml SF, 0,1 C. tr.PBMo/ml SF and QIAmp gel extraction kit®+ CTAB detecting 0,1 C. tr. -EB/ml SF, 1 C. tr.-PBMo/ml, respectively, allowed most sensitive detection of the organism in combination with the C. tr.- omp1-(152 bp) PCR
Sensitivity of Chlamydia-directed PCR and LCR testing for C. trachomatis EB DNA as a function of template preparation Highest sensitivity (0.1 C. trachomatis EB/ml SF) was achieved with the Qiaex II Gel Extraction Kit® + CTAB followed by alkaline lysis, Qiagen Tissue Kit® and QIAmp DNA Stool Kit®, which detected repeatedly 1 C. trachomatis EB/ml SF in combination with PCR 1
Summary
Polymerase chain reaction (PCR) and ligase chain reaction (LCR) are used in research for detection of Chlamydia trachomatis (C. tr.) in synovial fluid (SF). Chlamydia-induced arthritis (CIA) is the most frequent form of reactive arthritis (ReA) in western countries [1]. Immunofluorescence studies and RNA hybridization of synovial specimens were the first methods demonstrating intra-articularly persisting Chlamydia trachomatis [4,5]. Our results bp: base pairs; BSA: bovine serum albumin; CIA: chlamydia induced arthritis; EB: elementary bodies; IFU: infection forming units; LCR: ligase chain reaction; MOMP: major outer membrane protein; Omp-1: major outer membrane protein 1; PBMO: peripheral blood monocytes; PBS: phosphate buffered saline; PCR: polymerase chain reaction; ReA: reactive arthritis; SD: standard deviation; SF: synovial fluid
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