A highly sensitive, multiplex broad-spectrum PCR-DNA-enzyme immunoassay and reverse hybridization assay for rapid detection and identification of Chlamydia trachomatis serovars

J Mol Diagn. 2007 Nov;9(5):631-8. doi: 10.2353/jmoldx.2007.070011. Epub 2007 Sep 14.


Chlamydia trachomatis (Ct) comprises distinct serogroups and serovars. The present study evaluates a novel Ct amplification, detection, and genotyping method (Ct-DT assay). The Ct-DT amplification step is a multiplex broad-spectrum PCR for the cryptic plasmid and the VD2-region of ompl. The Ct-DT detection step involves a DNA enzyme immunoassay (DEIA) using probes for serogroups (group B, C, and intermediate) and the cryptic plasmid, permitting sensitive detection of 19 Ct serovars (A, B/Ba, C, D/Da, E, F, G/Ga, H, I/Ia, J, K, L1, L2/L2a, and L3) without any cross-reactivity with other Chlamydia species and pathogenic bacteria or commensal organisms of the genital tract. Ct-positive samples are analyzed by a nitrocellulose-based reverse hybridization assay (RHA) containing probes for the 19 different serovars and for the cryptic plasmid. The sensitivity of the PCR-DEIA on clinical specimen is equivalent to that of the Cobas TaqMan assay [kappa = 0.95 (95% confidence interval = 0.92 to 0.99)]. Using the RHA, 98% of the Ct-DT detection step-positive samples could be typed. Analysis of cervical swabs from Uganda and The Netherlands revealed that the most common serovars in Uganda are G/Ga (45%), E (21%), K (13%), and F (8%), and in The Netherlands serovars E (38%), F (23%), G/Ga (11%), and D/Da (7%) were most common. Thus, multiplex broad-spectrum PCR in combination with DEIA and RHA permits highly sensitive and specific detection and identification of Ct serovars.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Bacterial Typing Techniques
  • Base Sequence
  • Chlamydia trachomatis / classification
  • Chlamydia trachomatis / genetics*
  • Chlamydia trachomatis / isolation & purification*
  • DNA, Bacterial / analysis*
  • DNA, Bacterial / genetics*
  • Genotype
  • Immunoenzyme Techniques
  • Molecular Sequence Data
  • Nucleic Acid Hybridization / methods*
  • Polymerase Chain Reaction / methods*
  • Sequence Alignment
  • Vaginal Smears


  • DNA, Bacterial