Detection of Mycoplasma genitalium by PCR amplification of the 16S rRNA gene

Abstract

In order to develop a species-specific PCR for the detection of Mycoplasma genitalium, the sequence of 1,490 bases of the 16S rRNA gene was determined for M. genitalium G37 (type strain) and four Danish isolates of M. genitalium. The sequences of the four Danish strains, mutually different with respect to their MgPa gene, were 100% homologous, although they carried a single common base substitution compared to the type strain. Among members of the Mycoplasma pneumoniae phylogenetic cluster, M. genitalium showed the most-prominent homology to the 16S rRNA sequence of M. pneumoniae (98% homology). From regions showing the least homology to the M. pneumoniae 16S rRNA gene sequence, primers were chosen to amplify DNA from M. genitalium only. Two sets of primers were selected for their ability to detect <10 to 50 M. genitalium genome copies without cross-reactions with M. pneumoniae. The performance of these primers was compared to the performance of two pairs of primers amplifying parts of the MgPa adhesin gene; 1,030 randomly selected specimens submitted for Chlamydia trachomatis culture were screened with one of the 16S rRNA gene primer sets. A total of 41 specimens were found to be positive for this gene; 40 of these could be confirmed by one of the MgPa primer sets, whereas the other MgPa primer set detected only 21 positive specimens out of 40. These results indicate that estimates of the prevalence of M. genitalium in various populations using MgPa PCR primers could be incorrectly low if the PCR primers are located in variable regions of the MgPa gene.

FIG. 1.

LOD of MTP-based hybridization assay performed at 50°C with negative controls and various dilutions of M. genitalium amplicon. The biotinylated probe Mg16S-240 Bio was used at 10 pM. The negative control contained the IPC amplicon. Amplicons from M. pneumoniae were produced by low-stringency annealing of the primers.

FIG. 2.

Ethidium bromide-stained agarose gel showing the LOD of the M. genitalium 16S rRNA gene PCR and the effect of addition of the IPC and human DNA. Lane 1, 100-bp marker (Pharmacia); lane 2, ∼500 genome copies of M. genitalium DNA; lane 3, ∼50 genome copies of M. genitalium DNA; lane 4, ∼5 genome copies of M. genitalium DNA; lane 5, negative control; lanes 6 to 9, same as lanes 2 to 5 but with addition of IPC; lanes 10 to 13, same as lanes 2 to 5 but with addition of IPC and 10 μl of clinical urogenital specimen.