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Journal of Clinical Microbiology, January 2006, p. 206-213, Vol. 44, No. 1
0095-1137/06/$08.00+0     doi:10.1128/JCM.44.1.206-213.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Development and Validation of a Rotor-Gene Real-Time PCR Assay for Detection, Identification, and Quantification of Chlamydia trachomatis in a Single Reaction

Hamid Jalal,1* Hannah Stephen,1 Martin D. Curran,1 Janet Burton,1 Michelle Bradley,2 and Christopher Carne3

Clinical Microbiology & Public Health Laboratory, Box 236, Addenbrooke's Hospital, Hills Road, Cambridge CB2 2QW,1 Centre for Applied Medical Statistics, Institute of Public Health, University Forvie Site, Department of Public Health and Primary Care, Robison Way, Cambridge CB2 2SR,2 Department of Genitourinary Medicine, Clinic 1A, Box 38 Addenbrooke's Hospital, Hills Road, Cambridge, United Kingdom3

Received 29 July 2005/ Returned for modification 20 September 2005/ Accepted 19 October 2005

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.

* Corresponding author. Mailing address: Clinical Microbiology & Public Health Laboratory, Box 236, Addenbrooke's Hospital, Hills Road, Cambridge CB2 2QW, United Kingdom. Phone: 44-1223-257036. Fax: 44-1223-242775. E-mail: hamid.jalal{at}addenbrookes.nhs.uk.

Journal of Clinical Microbiology, January 2006, p. 206-213, Vol. 44, No. 1
0095-1137/06/$08.00+0     doi:10.1128/JCM.44.1.206-213.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

  • Petrovay, F., Balla, E., Nemeth, I., Gonczol, E. (2009). Genotyping of Chlamydia trachomatis from the endocervical specimens of high-risk women in Hungary. J Med Microbiol 58: 760-764 [Abstract] [Full Text]  
  • Jalal, H., Stephen, H., Alexander, S., Carne, C., Sonnex, C. (2007). Development of Real-Time PCR Assays for Genotyping of Chlamydia trachomatis. J. Clin. Microbiol. 45: 2649-2653 [Abstract] [Full Text]  
  • Jalal, H., Al-Suwaine, A., Stephen, H., Carne, C., Sonnex, C. (2007). Comparative performance of the Roche COBAS Amplicor assay and an in-house real-time PCR assay for diagnosis of Chlamydia trachomatis infection. J Med Microbiol 56: 320-322 [Abstract] [Full Text]  
  • Jaton, K., Bille, J., Greub, G. (2006). A novel real-time PCR to detect Chlamydia trachomatis in first-void urine or genital swabs.. J Med Microbiol 55: 1667-1674 [Abstract] [Full Text]  


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