JOURNAL OF CLINICAL MICROBIOLOGY, Mar. 2011, p. 1140–1142
Copyright © 2011, American Society for Microbiology. All Rights Reserved.
Vol. 49, No. 3
Comparison of Two Mycoplasma genitalium Real-Time
PCR Detection Methodologies?
Jimmy Twin,1,2* Nicole Taylor,1,2Suzanne M. Garland,1,2,3,4Jane S. Hocking,5Jennifer Walker,5
Catriona S. Bradshaw,6,7Christopher K. Fairley,5,6and Sepehr N. Tabrizi1,2,3
Department of Microbiology and Infectious Diseases, The Royal Women’s Hospital, Melbourne, Australia1;
Murdoch Childrens Research Institute, Melbourne, Australia2; Department of Obstetrics and Gynaecology,
University of Melbourne, Australia3; Department of Microbiology, The Royal Children’s Hospital,
Melbourne, Australia4; Melbourne School of Population Health, University of Melbourne,
Australia5; Melbourne Sexual Health Centre, Melbourne, Australia6; and Department of
Epidemiology and Preventive Medicine, Monash University, Melbourne, Australia7
Received 18 November 2010/Returned for modification 20 December 2010/Accepted 24 December 2010
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 My-
coplasma genitalium in the general population (3, 9, 12). In the
absence of adequate and reliable culture and approved com-
mercial assay techniques, most laboratories use in-house nu-
cleic acid amplification tests (NAATs) for detection of this
bacterium. Quantitative PCR (qPCR) assays have been de-
signed 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 ?5 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 ?10 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 multi-
plexed, and the authors recommended further testing in single-
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 LightCy-
cler 480 real-time PCR system (Roche Diagnostics), using
PCR conditions as described previously (8, 22). The MgPa
assay was able to detect ?6 copies/reaction of M. genitalium in
three replicate reactions, and the 16S rRNA gene assay de-
tected ?23 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 cy-
cle [Cq] ? 39.50; standard deviation [SD] ? 0.94), and the 16S
rRNA gene assay detected M. genitalium in seven (mean Cq ?
39.37; SD ? 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
initially screened for the 16S rRNA gene target; then samples
were stored at ?30°C for a median of 25 months (average ?
22.2; range ? 1 to 29) before subsequent testing for the MgPa
gene and retesting with the 16S RNA gene assay in cases where
the assays gave discordant results. Nine DNA samples tested
negative or yielded a low human ?-globin gene signal, indicat-
ing inadequate sampling, and were removed from subsequent
Also removed from the study were three DNA samples from
different patients in which M. genitalium was detected by the
16S rRNA assay (with 15, 68, and 129 copies per reaction;
* Corresponding author. Mailing address: Department of Microbi-
ology & Infectious Diseases, The Royal Women’s Hospital, Locked
Bag 300, Parkville, Vic. 3052, Australia. Phone: (61-3) 8345 3679. Fax:
(61-3) 9344 2713. E-mail: firstname.lastname@example.org.
?Published ahead of print on 5 January 2011.
TABLE 1. Clinical cohort screening sample results
No. of samples with MgPa result
Cq ? 40.48, 38.06, and 35.22, respectively) but which tested
negative with both the MgPa gene and 16S rRNA gene assay
after being kept in storage at ?30°C for 16 to 26 months.
Further repeated testing failed to generate PCR amplicons,
indicating that either multiple freeze-thaw cycles or extended
storage at ?30°C was likely to have resulted in degradation of
DNA samples. The potentially detrimental effect of freezing
on M. genitalium samples and DNA has been reported in other
studies (4, 13), and work in our own laboratory has shown
levels of M. genitalium DNA to significantly reduce after three
freeze-thaw cycles (data not shown). In future studies, freezing
of samples destined for M. genitalium testing is not recom-
mended, to avoid potential degradation.
Of the 40 samples found to be positive by one or both
methods (Table 1), 38 (88.4%) were positive by both assays
and 2 (4.7%) were positive by the MgPa assay only. Based on
these results, the sensitivity and specificity of the 16S rRNA
gene assay compared to those of the MgPa were 95.0% (95%
confidence interval [CI], 0.831 to 0.994) and 100%, respectively
(1). Kappa analysis of these two methods gave a score of 0.973
(95% CI, 0.936 to 1.010), indicating an “almost perfect” agree-
ment between these two assays (16). The two discordant sam-
ples in this study with M. genitalium detected only by the MgPa
assay were from the same patient but at different time periods
(recruitment and at a 12-month follow up), with concentrations
of 81 and 86 copies per reaction (Cq values ? 37.06 and 36.98,
Of the 38 samples that were positive for M. genitalium by
both assays, only one sample showed evidence of slight degra-
dation between retesting, showing a reduction from 411 copies
per reaction initially with the 16S rRNA gene assay (Cq ?
33.16) to fewer than 10 copies per reaction when retested with
both assays 26 months later (Cq ? 40.0 to 40.3). With the
remaining 37 samples, the calculated concentrations for the
MgPa assay ranged from 9 copies to 2.6 ? 105copies per
reaction (mean ? 3.0 ? 103; SD ? 5.5 ? 103), while results for
the 16S rRNA assay ranged from 34 copies to 1.7 ? 105copies
per reaction (mean ? 2.5 ? 103; SD ? 4.1 ? 103). Using a
paired t test, the concentrations given for each sample were not
statistically different overall (P ? 0.44). This was confirmed by
Deming regression analysis (6) giving a near-perfect line of
best fit (Fig. 1).
In summary, the MgPa and 16S rRNA gene assays appear
equally suitable for the detection of M. genitalium in vaginal
swabs. Further work is warranted to determine how effective
these methods are for other clinical specimen types and to
identify the reason(s) why two samples, from the same pa-
tient, gave rise to amplicons in the MgPa assay and not the
16S rRNA gene assay. These data will inform clinicians and
researchers regarding the comparative performances of the
two commonly used in-house assays for the detection of M.
genitalium and are of particular relevance as routine testing
and screening for the bacterium become increasingly com-
We thank the staff and students at the Women’s Centre for Infec-
tious Diseases (Royal Women’s Hospital, Victoria, Australia) for their
assistance in this study.
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FIG. 1. Scatter plot with Deming line of best fit. The log10trans-
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VOL. 49, 2011NOTES1141
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1142 NOTESJ. CLIN. MICROBIOL.