RESUMO
Introduction. Chlamydia trachomatis (C. trachomatis, CT) is an obligatory intracellular bacterium that causes urogenital tract infections and leads to severe reproductive consequences. Therefore, a rapid and accurate detection method with high sensitivity and specificity is an urgent requirement for the routine diagnosis of C. trachomatis infections.Aim. In this study, we aimed to develop a multiplex quantitative real-time PCR (qPCR) assay based on two target regions for accurate detection of C. trachomatis in urogenital tract infections.Methodology. Primers and probes based on the conserved regions of the cryptic plasmid and 23S rRNA gene were designed. Then, two qPCR assays were established to screen for the optimal probe and primers for each of the two target regions. Subsequently, the multiplex qPCR method was developed and optimized. For the diagnostic efficiency evaluation, 1284 urogenital specimens were tested by the newly developed multiplex qPCR method, an immunological assay and a singleplex qPCR assay widely used in hospitals.Results. The multiplex qPCR method could amplify both target regions in the range of 1.0×102-1.0×108 copies ml-1 with a strong linear relationship, and lower limits of detection (LODs) for both targets reached 2 copies PCR-1. For the multiplex qPCR method, the diagnostic sensitivity and specificity was 100.0â% (134/134) and 99.3â% (1142/1150), respectively. For the singleplex qPCR assay, the diagnostic sensitivity and specificity was 88.8â% (119/134) and 100.0â% (1150/1150), respectively. For the immunological assay, the diagnostic sensitivity and specificity was 47.0â% (63/134) and 100.0â% (1150/1150), respectively.Conclusion. In this study, a multiplex qPCR assay with high sensitivity and specificity for rapid (≤2.0 h) and accurate diagnosis of C. trachomatis was developed. The qPCR assay has the potential to be used as a routine diagnostic method in clinical microbiology laboratories.
