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Journal of Clinical Microbiology, July 2005, p. 3121-3128, Vol. 43, No. 7
0095-1137/05/$08.00+0     doi:10.1128/JCM.43.7.3121-3128.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Development of a Quantitative Real-Time PCR Assay for Detection of Mycoplasma genitalium

Helle Friis Svenstrup,1* Jørgen Skov Jensen,2 Eva Björnelius,3 Peter Lidbrink,3 Svend Birkelund,1 and Gunna Christiansen1

Department of Medical Microbiology and Immunology, The Bartholin Building, Aarhus University, DK-8000 Aarhus C, Denmark,1 Mycoplasma Laboratory, Statens Serum Institut, DK-2300 Copenhagen S, Denmark,2 Department of Dermatovenereology, Huddinge University Hospital, Karolinska Institutet, S-14 186 Huddinge, Sweden3

Received 31 January 2005/ Returned for modification 3 March 2005/ Accepted 9 March 2005

Mycoplasma genitalium is known to cause nonchlamydial, nongonococcal urethritis in men and to be associated with pelvic inflammatory disease in women. Specific and sensitive PCR methods are needed for diagnosis of this bacterium because it is very difficult to culture from patient samples. To determine the bacterial load in patients' specimens, a quantitative real-time LightCycler PCR was developed. The housekeeping gene gap encoding glyceraldehyde-3-phosphate dehydrogenase was chosen as the target gene. The assay could consistently detect five genome copies per reaction. To evaluate the PCR, we tested 246 selected urethral swab samples from men attending a clinic for sexually transmitted diseases. Eighty-two of the samples were found positive for M. genitalium by a conventional 16S rRNA gene PCR assay, whereas 164 samples were randomly chosen among those tested negative. Of the positive samples, 78 (95.1%) were found positive, whereas 6 (3.7%) of the negatives were found positive by the LightCycler assay. The patient samples were also tested with a quantitative TaqMan assay, and the bacterial load was compared to the LightCycler results. A good linear correlation between the LightCycler and the TaqMan assays was found with a correlation coefficient of 0.89 and a slope of 0.99. Significantly more M. genitalium-positive men had urethritis, discharge, and dysuria than had M. genitalium-negative men. The M. genitalium DNA load in samples from patients with urethritis was significantly higher than in samples from those without (61 and 2.9 copies/µl, respectively [P = 0.0005]). This assay may prove useful in the monitoring of treatment and for optimizing sample preparation methods.

* Corresponding author. Mailing address: Department of Medical Microbiology and Immunology, The Bartholin Building, Aarhus University, DK-8000 Aarhus C, Denmark. Phone: 45 8942 1746. Fax: 45 8619 6128. E-mail: hellef{at}medmicro.au.dk.

Journal of Clinical Microbiology, July 2005, p. 3121-3128, Vol. 43, No. 7
0095-1137/05/$08.00+0     doi:10.1128/JCM.43.7.3121-3128.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.



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