Comparison of Two Mycoplasma genitalium Real-Time PCR Detection Methodologies

Abstract

Established in-house quantitative PCR (qPCR) assays to detect the Mycoplasma genitalium adhesion protein (MgPa) and the 16S rRNA gene were found to be comparable for screening purposes, with a kappa value of 0.97 (95% confidence interval [CI], 0.94 to 1.01) and no difference in bacterial load quantified (P ‫؍‬ 0.4399).Limited knowledge exists regarding the epidemiology of Mycoplasma genitalium in the general population (3,9,12). In the absence of adequate and reliable culture and approved commercial assay techniques, most laboratories use in-house nucleic acid amplification tests (NAATs) for detection of this bacterium. Quantitative PCR (qPCR) assays have been designed for a variety of M. genitalium targets (2,5,7,8,14,15,17,18,20,22), though the most cited qPCR assays are the one described by Jensen et al., which targets a 78-bp region of the M. genitalium adhesion protein (MgPa) and has a reported sensitivity of Ͻ5 copies per reaction (14), and an assay by Yoshida et al. (22), which targets a 517-bp region of the 16S rRNA gene with a sensitivity of Ͼ10 copies per reaction. Both of these targets are present as a single copy in the M. genitalium genome (10). Hardick et al. described a multiplex assay that incorporated both the MgPa and 16S rRNA gene qPCR assays (11) and found that the 16S rRNA target did not detect 59 of 607 samples (9.7%) in which MgPa was detected. Lack of detection by the 16S rRNA gene component could possibly be attributed to competition when the two targets were multiplexed, and the authors recommended further testing in singleplex reactions.To investigate the issue of varying sensitivities between the MgPa and 16S rRNA gene assays, an initial experiment was carried out to determine the detection limit of each assay. A clinical sample equivalent to 1,200 copies/l of M. genitalium was diluted 1:4 to extinction and run in triplicate. Each assay consisted of 5 l template in a 20-l reaction on the LightCycler 480 real-time PCR system (Roche Diagnostics), using PCR conditions as described previously (8, 22). The MgPa assay was able to detect Ն6 copies/reaction of M. genitalium in three replicate reactions, and the 16S rRNA gene assay detected Ն23 copies per reaction. Both assays were able to detect M. genitalium down to a single copy although in only one of three replicate reactions each. Further analysis on a separate clinical sample diluted to approximately six copies per reaction was carried out with 12 replicates. The MgPa assay detected M. genitalium in eight of these reactions (mean quantification cycle [Cq] ϭ 39.50; standard deviation [SD] ϭ 0.94), and the 16S rRNA gene assay detected M. genitalium in seven (mean Cq ϭ 39.37; SD ϭ 0.80).Testing was then carried out on 845 self-collected vaginal swab samples (from 761 individuals) obtained as part of the Chlamydia Incidence and Re-Infection Rates Study (CIRIS) (21), which consisted of specimens collected at the recruitment and at a 12-month follow-up. Sample processing and DNA extraction were as described previously (19). Each sample was in...

Mycoplasma genitalium and Cancer: A Brief Review

“…One of the major reasons for little studies in this area might be fastidious nature of M genitalium to culture that limited direct detection and required researchers to develop other methods. Nucleic acid amplification-based methods have widely used for detection of M genitalium in urogenital specimens (de Barbeyrac et al, 1993;Jurstrand et al, 2005;Edberg et al, 2008;Ouzounova-Raykova et al, 2011;Twin et al, 2011), so it seems, this techniques and ELISA which has used to show antibody status against M genitalium in patient sera (Baseman et al, 2004;Svenstrup et al, 2006) could be useful tools in epidemiological studies. Immunohistochemistry (IHC) has been used to detection of Mycoplasma species in clinical tissue samples (Adegboye et al, 1995;Moorkamp et al, 2010;Yang et al, 2010a;2010b) but, this method has not developed for M genitalium, although polyclonal and monoclonal antibodies (as main tools for Immunohistochemistry and other immunoassays methods) against major antigens of M genitalium have been produced (Svenstrup et al, 2002;Burgos et al, 2006;Zarei et al, 2011).…”

Isolation of Mycoplasma genitalium from endocervical swabs of infertile women

“…Mycoplasma genitalium was detected in mid-trimester of women delivering preterm,6 and among women with tubal factor infertility 7. Immunological assays cannot reliably detect the presence of M. genitalium 8. Currently, several tests are being used to detect Mycoplasma infections including nucleic acid amplification testing (NAATs), and a quantitative real-time Light Cycler polymerase chain reaction (PCR) assay which targets the gene gap encoding glyceraldehyde-3-phosphate dehydrogenase 9.…”

Evaluation of the Hologic Panther Transcription-Mediated Amplification Assay for Detection of Mycoplasma genitalium

“…Diagnosis relies on nucleic acid amplification tests (NAAT); however, as there have been limited commercial assays available, most laboratories have utilized in-house NAATs, utilizing quantitative PCR (qPCR) assays with various targets (15)(16)(17)(18)(19)(20)(21). For some time, a research-use-only transcription-mediated amplification assay for detection of M. genitalium (MG-TMA) has been available from Hologic (Bedford, MA, USA) for use on either the manual or automated TIGRIS DTS system; however, this has recently been introduced onto the Panther platform utilizing the open-channel software feature, with the manufacturer package insert indicating a sensitivity of 0.01 CFU/ml (equivalent to 0.004 copies per reaction).…”

“…The resuspended pellet was subsequently extracted using MagNA Pure 96 (Roche Diagnostics GmbH, Penzberg, Germany) and a DNA and viral nucleic acid small-volume kit (Pathogen Universal 200 protocol). Extracted DNA was eluted into a final volume of 100 l: 5-l aliquots were utilized in two qPCR assays targeting the 16S rRNA gene (20) and the MgPa gene (18). Both qPCR assays were run on a Roche LC480 real-time PCR instrument using Sensi-FAST Probe No-ROX chemistry (Bioline, Alexandria, NSW, Australia).…”