A new real-time quantitative TaqMan PCR to detect Chlamydia trachomatis DNA in urine and in urogenital swabs
Abstract number: p957
Jaton K., Bille J., André C., Greub G.
Chlamydia trachomatis infection is the most frequent bacterial sexually transmitted disease. Urogenital infections are often asymptomatic and, when remaining untreated, they may lead to severe late complications in women, including infertility and extra-uterine pregnancy. Screening for Chlamydia trachomatis infections may be performed on urines and urogenital swabs using molecular detection techniques. However, commercially available methods remain expensive and are associated with large laboratory workload. To reduce costs and increase throughput, we developed a C. trachomatis specific real-time quantitative TaqMan PCR (q-PCR).
Material and methods:
A q-PCR that may be used on ABI Prism 7900R Sequence System (Applied Biosystem) was developed. This system was coupled to a liquid handling unit (TecanR), allowing automatic pipetting and use of 384 wells micro plates. DNA was extracted with the Magna-PureR (Roche), and q-PCR was performed using 5 ml DNA and 15 ml PCR mixture. Primers and probe were selected from highly conserved sequence of the cryptic plasmid. This new q-PCR was compared to the commercial Cobas AmplicorR (C-PCR) on a total of 646 specimens taken from 564 patients (297 urogenital swabs and 349 urine samples).
The q-PCR exhibited the same analytical sensitivity as C-PCR. The analytical specificity of q-PCR was excellent with no signal detected in presence of bacterial DNA of 7 others Chlamydia species and of 15 other bacterial species potentially present in urogenital samples. No inhibition of PCR reaction was observed with q-PCR whereas 19 out of 646 samples (2.9%) were inhibited with the C-PCR. Comparison of the remaining 627 specimens showed an overall agreement of 99.7% between C-PCR and q-PCR (95 +/+, 530 -/-, 2 ±, 0 ±). Considering the C-PCR as gold standard, the specificity was 100% (530/530) and the sensitivity of 98% (95/97). The 2 discrepant results were re-investigated in a second run of C-PCR and q-PCR. One was found strongly positive with the 2 methods. The other was found negative in a second run of Cobas and once positive by q-PCR when 5 additional replicates were done, suggesting that there is a very low amount of DNA in this sample.
The q-PCR is a specific method that exhibits a similar sensitivity than C-PCR for the detection of C. trachomatis. Given its high throughput potential and the low costs of q-PCR reagents (about 2 $ per sample), it may contribute to future large-screening program.
|Session name:||XXIst ISTH Congress|
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