Development of a rotor-gene real-time PCR assay for the detection and quantification of Mycoplasma genitalium

We developed and validated a real-time quantitative polymerase chain reaction (qPCR) assay to determine . Mycoplasma genitalium bacterial load in endocervical swabs, based on amplification of the . pdhD gene which encodes dihydrolipoamide dehydrogenase, using the Rotor-Gene platform. We first determined the qPCR assay sensitivity, limit of detection, reproducibility and specificity, and then determined the ability of the qPCR assay to quantify . M. genitalium in stored endocervical specimens collected from Zimbabwean women participating in clinical research undertaken between 1999 and 2007. The qPCR assay had a detection limit of 300 genome copies/mL and demonstrated low intra- and inter-assay variability. The assay was specific for . M. genitalium DNA and did not amplify the DNA from other mycoplasma and ureaplasma species. We quantified . M. genitalium in 119 of 1600 endocervical swabs that tested positive for . M. genitalium using the commercial Sacace . M. genitalium real-time PCR, as well as 156 randomly selected swabs that were negative for . M. genitalium by the same assay. The . M. genitalium loads ranged between <. 300 and 3,240,000 copies/mL. Overall, the qPCR assay demonstrated good range of detection, reproducibility and specificity and can be used for both qualitative and quantitative analyses of . M. genitalium in endocervical specimens and potentially other genital specimens. © 2011 Elsevier B.V..