Lien DOI – http://dx.doi.org/10.7124/bc.000976
Biopolymers and Cell. 2018. Vol. 34. N 2. P 117–128
The first-generation tests targeting a cryptic plasmid for theC. 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 plasmid-
based 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 introduc-
tion of a Taqman probe (essential for the real-time PCR) scales the assay down to C. trachoma-
tis. At the same time, our plasmid PCR is specific toC. 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.