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. Dragon Magazine 390 Pdf Printer.

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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. Chlamydia trachomatis is a bacterial infection of global public health significance (). It is an intracellular pathogen with 15 serotypes. It causes trachoma (serotypes A, B, B 1, and C), lymphogranuloma venereum (serotypes L 1, L 2, and L 3), and oculogenital infections (serotypes D to K). There are many highly conserved nucleotide sequences in different chromosomal genes and in the cryptic plasmid (CP) of C. These sequences are used as targets for nucleic acid amplification tests (NAATs).

NAATs have surpassed cell culture and antigen detection for the diagnosis of C. Trachomatis infections due to their enhanced sensitivities. A large number of in-house NAATs for the detection of C.

Trachomatis have been reported (). The majority of these are gel based. A few non-gel-based NAATs have also been described, i.e., a Qβ replicase-amplified assay () and PCR-enzyme immunoassays (,, ). All previously reported in-house NAATs are manual and use open systems with separate steps for amplification and amplicon analysis. Due to the risk of contamination and a lack of automation, these assays are inappropriate for diagnostic laboratories with high workloads. Real-time PCR is performed in a closed system, amplification and detection of target are done in a single step, and there is the potential for automation. Real-time PCR has been used for pharmacodynamic studies for C.

Trachomatis (, ). Two real-time PCR assays for the detection of C. Trachomatis have also been described (, ). Both these assays used the SYBR green technology in the LightCycler system (Roche Molecular Systems, Inc., Pleasanton, CA) and were used for the qualitative detection of C. Roche Molecular Systems, GEN-PROBE Inc.

(San Diego, CA), BD Diagnostics (Sparks, MD), and artus GmbH (Hamburg, Germany) are the four major commercial companies which supply NAATs for the detection of C. Trachomatis in Europe. At present, artus GmbH is the only commercial company that provides an assay for the quantification of C. Trachomatis in clinical samples. A single gene of C. Trachomatis per amplification reaction was targeted in all of the in-house and commercial assays mentioned above. The targets of amplification were the CP, the major outer membrane protein (MOMP) gene, the 23S rRNA gene, and the cysteine-rich outer membrane protein gene ().

In the present study, we report on a multitarget real-time PCR (MTR-PCR) assay for the detection, identification, and quantification of C. Trachomatis in a single reaction. Three targets of amplification were used in this assay: the cryptic plasmid for the qualitative detection, the MOMP gene for the quantification of C. Trachomatis DNA, and an internal control (IC) for the detection of inhibitors of amplification. None of the previously reported in-house or commercial assays have achieved these characteristics in a single reaction.