Quantitative detection of Chlamydia spp. by fluorescent PCR in the LightCycler.
Quantitative detection of intracellular bacteria of the genus Chlamydia by the standard cell culture method is cumbersome and operator dependent. As an alternative, we adapted hot-start PCR to the glass capillary quantitative PCR format of the LightCycler. The optimized PCR was consistently more efficient than commercially available pre-assembled PCRs. Detection by quantitative PCR of as few as single copies of DNA of Chlamydia spp. was accomplished by SYBR Green fluorescence of the dsDNA product and by fluorescence resonance energy transfer (FRET) hybridization probes. The PCRs were 15-fold more sensitive than the cell culture quantitative assay of C. psittaci B577 infectious stock. The number of chlamydial genomes detected by C. psittaci B577 FRET PCR correlated well with cell culture determination of inclusion forming units (IFUs) (r = 0.96, P < 0.0008). When infected tissue samples were analyzed by cell culture and PCR, the correlation coefficient between IFUs and chlamydial genomes was higher with C. psittaci B577 FRET PCR (r = 0.90, P < 0.0004) than with Chlamydia omp1 SYBR Green PCR (r = 0.85, P < 0.002).
Huang
J
J
College of Veterinary Medicine, Auburn University, Auburn, AL, USA.
DeGraves
F J
FJ
Gao
D
D
Feng
P
P
Schlapp
T
T
Kaltenboeck
B
B
D001425:Bacterial Outer Membrane Proteins / Q000235:genetics; D001483:Base Sequence; D052160:Benzothiazoles; D002689:Chlamydia / Q000235:genetics*; D004269:DNA, Bacterial / Q000235:genetics; D003959:Diamines; D005434:Flow Cytometry / Q000295:instrumentation; D005456:Fluorescent Dyes; D005470:Fluorometry / Q000295:instrumentation; D008969:Molecular Sequence Data; D009930:Organic Chemicals; D016133:Polymerase Chain Reaction / Q000379:methods*; D018272:Porins; D011804:Quinolines; D015203:Reproducibility of Results
D001425:Bacterial Outer Membrane Proteins; D052160:Benzothiazoles; D004269:DNA, Bacterial; D003959:Diamines; D005456:Fluorescent Dyes; D009930:Organic Chemicals; D018272:Porins; D011804:Quinolines; C071677:omp1 protein, Chlamydia trachomatis; C098022:SYBR Green I