Quantitative detection of Chlamydia spp. by fluorescent PCR in the LightCycler (R)

Quantitative detection of intracellular bacteria of the genus Chlamydia by the standard cell culture method is cumbersome and operator dependent. As a n alternative, ire adapted hot-start PCR to the glass capillary quantitativ e PCR format of the Light-Cycler(R). The optimized PCR was consistently I,r are efficient than commercially available pre-assembled PCRs. Detection by quantitative PCR of as few as single copies of DNA of Chlamydia spp. was ac complished by SYBR(R) Green fluorescence of the dsDNA product and by fluore scence resonance energy transfer (FRET) hybridization probes. The PCRs were 15-fold more sensitive than the cell culture quantitative assay of C. psit taci 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 = 0.8 5, P < 0.002).