Detection of Chlamydia trachomatis-DNA in synovial fluid: evaluation of the sensitivity of different DNA extraction methods and amplification systems.
BACKGROUND
Polymerase 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.


METHODS
SF 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).


RESULTS
In 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 degrees C for 4 months by at least one log phase.


CONCLUSIONS
The 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.
Freise
Julia
J
Division of Pneumology, Hannover Medical School, Carl-Neuberg Strasse 1, Hannover, 30625, Germany. juliafreise@hotmail.com
Bernau
Iris
I
Meier
Sabine
S
Zeidler
Henning
H
Kuipers
Jens G
JG
D001170:Arthritis, Infectious / Q000175:diagnosis*; D002690:Chlamydia Infections / Q000175:diagnosis*; D002692:Chlamydia trachomatis; D004269:DNA, Bacterial / Q000302:isolation & purification*; D006801:Humans; D022521:Ligase Chain Reaction / Q000379:methods / Q000592:standards*; D016133:Polymerase Chain Reaction / Q000379:methods / Q000592:standards*; D012680:Sensitivity and Specificity; D013582:Synovial Fluid / Q000382:microbiology*
D004269:DNA, Bacterial