Development and validation of a rotor-gene real-time PCR assay for detection, identification, and quantification of Chlamydia trachomatis in a single reaction

Abstract

A multitarget real-time PCR (MRT-PCR) for detection of Chlamydia trachomatis DNA was developed and validated. There were three targets for amplification in a single reaction: the cryptic plasmid (CP), the major outer membrane protein (MOMP) gene, and an internal control. The assay had the following characteristics: (i) detection and confirmation of the presence of C. trachomatis DNA in a single reaction, (ii) detection of all genovars of C. trachomatis without any cross-reactivity with pathogenic bacteria or commensal organisms of the oropharynx and genital tract, (iii) a 95% probability of detection with three copies of MOMP and one copy of CP per reaction mixture, (iv) identification of the inhibition of amplification, (v) a quantitative dynamic range of 25 to 250,000 genome copies per reaction mixture, (vi) high intra- and interassay reproducibilities, and (vii) correct identification of all samples in the validation panel. There were 146 COBAS Amplicor PCR (Amplicor PCR)-positive samples and 122 Amplicor PCR-negative samples in the panel. MRT-PCR detected CP DNA alone in 6 (4%) Amplicor PCR-positive samples and both CP and MOMP DNAs in 140 (96%) of 146 Amplicor PCR-positive samples. The quantity of MOMP DNA in 95 (68%) of 140 samples was within the dynamic range of the assay. The median C. trachomatis load in these samples was 321 genome copies per reaction mixture (range, 26 to 40,137 genome copies per reaction mixture). Due to the inclusion of two different C. trachomatis-specific targets, the assay confirmed 259 (97%) of 268 results in a single reaction. This assay could be used in the qualitative format for the routine detection of C. trachomatis and in the quantitative format for study of the pathogenesis of C. trachomatis-associated diseases. The assay demonstrated the potential to eliminate the need for confirmatory testing in almost all samples, thus reducing the turnaround time and the workload.

FIG. 1.

Algorithm for interpretation of assay results. Plasmid, MOMP, and IC represent the three components of the assay. *, high chlamydia genome copy number in the reaction may suppress amplification of the IC.

FIG. 2.

Intra- and interassay reproducibilities. Ten replicates of each of 10-fold serial dilutions of C. trachomatis DNA were included per run. Color code: blue, 250,000 genome copies per reaction mixture; orange, 25,000 genome copies per reaction mixture; purple, 2,500 genome copies per reaction mixture; green, 250 genome copies per reaction mixture; red, 25 genome copies per reaction mixture.

FIG. 3.

Linearity of the assay. Data are from three 10-fold dilution panels of C. trachomatis (CT) DNA (10 replicates each) ranging from 25 to 250,000 genome copies per reaction mixture.