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.
Centre for HIV and Sexually Transmitted Infections, National Institute for Communicable Diseases, National Health Laboratory Service, Johannesburg, South Africa. etiennem@nicd.ac.za
Etienne E
EE
Müller
Johanna M E
JM
Venter
Mahlape P
MP
Magooa
Charles
C
Morrison
David A
DA
Lewis
Sue Napierala
SN
Mavedzenge
D058491:Bacterial Load / Q000379:methods*; D002584:Cervix Uteri / Q000382:microbiology; D004269:DNA, Bacterial / Q000032:analysis* / Q000235:genetics; D005260:Female; D006801:Humans; D009175:Mycoplasma Infections / Q000382:microbiology; D045704:Mycoplasma genitalium / Q000235:genetics / Q000302:isolation & purification*; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D015030:Zimbabwe
D004269:DNA, Bacterial