A dual-target strategy for the detection of Chlamydia trachomatis by real-time PCR

Abstract

The first-generation tests targeting a cryptic plasmid for the C. trachomatis diagnostics showed a relatively high sensitivity; however their usefulness has recently been compromised by the discovery of C. trachomatis strains lacking the target DNA segment (e.g. the "Swedish" variant) or variants bearing no plasmid at all and thus escaping the diagnostics. Aim. We propose the addition of a C. trachomatis chromosome gene as a PCR target to back up plasmidbased assays and enhance the overall efficiency of diagnostics. Methods. Two multiplexed PCRs were set up to target C. trachomatis cryptic plasmid and the 16s rRNA gene. The 16s rRNA primers produce PCR signal from a range of Chlamydia species whereas the introduction of a Taqman probe (essential for the real-time PCR) scales the assay down to C. trachomatis. At the same time, our plasmid PCR is specific to C. trachomatis exclusively. Results. The sensitivity of plasmid and 16s rRNA PCRs ranged between one to ten genome-equivalents per reaction (geq/rxn) whereas the efficiency was always ~100%. Multiplexing did not reduce the analytical sensitivity. Addition of DNA prepared from clinical specimens to the reaction mix did not affect PCR with pure C. trachomatis DNA further demonstrating the robustness of this system. The kinetics of the two reactions was compared in 49 DNA samples prepared from C. trachomatis-positive swabs. In 45, reactions showed a good correlation in the threshold cycle of amplification Cq, the main analytical parameter of real-time PCR. Conclusions. The simultaneous detection of chromosomal and plasmid targets in the multiplex PCR offers a high sensitivity and is particularly advantageous for specimens where the plasmid might be lost due to DNA degradation or counter-selection after treatment. The dual PCR strategy constitute the core of a diagnostic test for both in-house and commercial use.

Chlamydia trachomatis intra-bacterial and total plasmid copy number in clinical urogenital samples

“…These findings demonstrate an additional reason for the use of the CT plasmid in commercial assays 20 . It has been known that new genetic variants might be missed by single-target assay, as demonstrated by the Swedish new variant CT 21,22 , and since then it is recommended to use dual-target assays [23][24][25] . Yet, of the five FDA approved and commercially available NAATS (Roche Diagnostics, Becton Dickinson and Company, Hologic/ Gen-Probe Inc., Abbott Laboratories, and Cepheid), one detects only a chromosomal target, two only plasmid targets, one detects rRNA and only one detects both 26 .…”