Ultra-rapid detection of Chlamydia trachomatis by real-time PCR in the LightCycler using SYBR green technology or 5'-nuclease probes.
Chlamydia trachomatis infections are among the most common sexually transmitted diseases and of great epidemiological importance world-wide. Identification of this pathogen can be difficult, and it is highly desirable to have a rapid and accurate nucleic acid based detection method. Several commercial PCR test systems are available (e.g. CobasAmplicor, Roche, Mannheim, Germany) but they require post-amplification detection by hybridization resulting in extended work-up time and possible cross-contamination. The objective of our study was to develop a routine diagnostic method for the sensitive, specific and rapid detection of C. trachomatis. The obvious choice is real-time PCR without any post-amplification procedures. The dye SYBR Green I (intercalating in dsDNA) provides a simple and fast real-time PCR in the LightCycler. Specific primer design combined with melting curve analysis allows a reliable and sensitive identification of C. trachomatis. In addition, a new commercial real-time PCR system (RealArt C. trachomatis LC PCR Reagents, artus, Hamburg, Germany) was evaluated, that combines sequence-specific primers and fluorescence-labelled (FRET) 5'-nuclease probes. An internal control integrated in this system detects false negative results and erroneous PCR conditions. All results were compared with the corresponding data from an analysis using the CobasAmplicor system (Roche). (Clin
artus GmbhH, Hamburg, Germany.
Meike
M
Eickhoff
Thomas
T
Laue
Tobias
T
Ruckes
Sven O
SO
Cramer
Guido
G
Krupp
Carsten
C
Tiemann
D000328:Adult; D052160:Benzothiazoles; D002690:Chlamydia Infections / Q000175:diagnosis*; D002692:Chlamydia trachomatis / Q000235:genetics / Q000302:isolation & purification*; D015342:DNA Probes / Q000737:chemistry; D004269:DNA, Bacterial / Q000032:analysis; D003959:Diamines; D005260:Female; D005453:Fluorescence; D005456:Fluorescent Dyes; D006801:Humans; D008297:Male; D009930:Organic Chemicals; D011804:Quinolines; D011933:Reagent Kits, Diagnostic; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000295:instrumentation / Q000379:methods*; D012680:Sensitivity and Specificity
D052160:Benzothiazoles; D015342:DNA Probes; D004269:DNA, Bacterial; D003959:Diamines; D005456:Fluorescent Dyes; D009930:Organic Chemicals; D011804:Quinolines; D011933:Reagent Kits, Diagnostic; C098022:SYBR Green I