Development of a quantitative real-time PCR assay for detection of Mycoplasma genitalium

Abstract

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/microl, respectively [P = 0.0005]). This assay may prove useful in the monitoring of treatment and for optimizing sample preparation methods.

FIG. 1.

Sequence of gap corresponding to the amplicon of the M. genitalium LightCycler assay. The gap sequence of M. genitalium (upper sequence) was compared to the homologous gap sequence of M. pneumoniae (lower sequence) by use of the GCG program Gap alignment (BioBase; www.biobase.dk). The selected primers and probes in the M. genitalium gap sequence are underlined. Primers were designed to differ from the M. pneumoniae sequences, especially at the 3′ ends. The probes were located on the same strand as the forward primer; to ensure good quantitative detection, they were placed closest to the reverse primer. Furthermore, 12 mismatches to M. pneumoniae were present in the probe region. In one M. genitalium-positive patient sample, a sequence analysis of the PCR product showed that guanine was replaced by adenine in position 682, as marked on the figure.

FIG. 2.

(Top) Fluorescence curves of the standard dilution series. Fluorescence curves from 10⁵ copies/μl down to 1 copy/μl are shown. Two negative controls are also included: one without DNA and one with M. pneumoniae DNA (10⁵ copies/μl). F2 is the channel at which the fluorescence is measured at 640 nm. F1 is the channel at which the fluorescence is measured at 530 nm. (Bottom) Standard curve used to calculate the concentration of M. genitalium DNA in unknown samples. The PCR cycle number is plotted against the log concentration of the standard dilutions. The linear regression coefficient is close to 1, and the 10^(−1/slope) value is ≈2.0, indicating a 100% efficiency of the PCR.

FIG. 3.

The four-parametric sigmoid model (described by equation 1) used on fluorescence data from the spiking assay. In this example, the standard curve of 10² copies/μl is shown. The background fluorescence is designated y₀, a is the height of the curve, x₀ is the first derivative maximum of the function, and b is the slope of the curve.

FIG. 4.

The LightCycler assay was compared to the TaqMan assay (12). Seventy-eight patient samples were found positive and quantified by both PCR assays, and the results were plotted against each other. The linear regression line is shown as a solid line, whereas the expected 1:1 line is shown as a dotted line. Note the high linear correlation coefficient of 0.89 and the proximity to the 1:1 line. One specimen was negative by both assays; an additional three specimens were negative by the LightCycler assay but positive by the TaqMan assay (not shown).