Rapid detection of Chlamydia trachomatis and typing of the Lymphogranuloma venereum associated L-Serovars by TaqMan PCR

Abstract

Background: Infection due to Chlamydia trachomatis is the most common sexually transmitted bacterial disease of global health significance, and especially the L-serovars causing lymphogranuloma venereum are increasingly being found in Europe in men who have sex with men.

Results: The design and evaluation of a rapid, multiplex, real-time PCR targeting the major outer membrane protein (omp-1) -gene and a L-serovar-specific region of the polymorphic protein H (pmp-H) -gene for the detection of Chlamydia trachomatis is reported here. The PCR takes place as a single reaction with an internal control. For L1-, L2- and L3-serovar differentiation a second set of real-time PCRs was evaluated based on the amplification of serovar-specific omp-1-regions. The detection limit of each real-time PCR, multiplexed or not, was 50 genome copies per reaction with an efficiency ranging from 90,5-95,2%. In a retrospective analysis of 50 ocular, rectal and urogenital specimens formerly tested to be positive for C. trachomatis we identified six L2-serovars in rectal specimens of HIV-positive men, one in a double-infection with L3, and one L2 in a urethral specimen of an HIV-negative male.

Conclusion: This unique real-time PCR is specific and convenient for the rapid routine-diagnostic detection of lymphogranuloma venereum-associated L-serovars and enables the subsequent differentiation of L1, L2 and L3 for epidemiologic studies.

Figure 1

Alignments of the omp-1- (a) and pmp-H-gene regions (b) by the use of different C. trachomatis serovars A to L. The corresponding accession numbers are given in Figure 3 (omp-1) and in Material and Methods (pmp-H). Identical nucleotides are boxed, the binding sites of primers and probes are marked above. *, difference in the Ct-R1 and -R2 primers.

Figure 2

Amplification charts and standard curves for the CT/LGV multiplexed real-time PCR. The linear range of the assay was determined using duplicates of 1.25 × [10⁹, 10⁷, 10⁵, 10³, 10², 10¹] copies of each cloned amplicon. The threshold values (C_t) were plotted against the corresponding copy numbers, and the efficiency, slope and linear regression correlation (r²) were calculated for each reaction by the Biorad IQ5 software.

Figure 3

(a) Phylogenetic tree of the omp-1-gene region of 20 C. trachomatis serovars targeted in the L1-, L2- and L3-Real-time PCR. (b) Alignments of the respective omp-1-sequences of the serovars A to L grouped in the L1-, L2- or L3-cluster. The accession numbers of the serovars are indicated. Identical nucleotides are boxed, the binding sites of primers and probes are marked above.