Taking advantage of the genomic sequence data now available for all 14 Ureaplasma serovars, our multiplex real-time PCR assay for the simultaneous detection and discrimination of the two Ureaplasma species targets two conserved gene sequences, each present only in U. parvum or U. urealyticum. This differs from previously reported assays for Ureaplasma species identification that targeted small differences within genes conserved in both species. The specificity of this assay was ensured computationally and experimentally verified by BLAST analysis and running the assay against a broad range of other microorganisms.
Compared to culture and traditional PCR, our findings using a single-step multiplex assay format agreed with those of Cao et al. (6
), who found the real-time PCR to be the most sensitive in the detection of Ureaplasma
species. The detection threshold of our assays is well below the level necessary to detect ureaplasmas in clinical specimens, and our multiplex PCR detected Ureaplasma
spp. in 15.2% more clinical specimens than did culture in a direct comparison using optimum cultivation methods developed and validated in our laboratory over several years (34
). Even though culture is often considered the reference method for the detection of ureaplasmas in clinical specimens, improved PCR assays may be inherently more sensitive and capable of detecting the organisms in very low numbers. Thus, the PCR-positive and culture-negative specimens we encountered are likely to be true positives rather than false negatives. Whether a PCR assay is more sensitive than culture for the detection of ureaplasmas in clinical specimens depends on the PCR target, the assay conditions, and the method of culture used. Numerous studies have shown clearly that PCR assays can be superior to culture (1
). However, even though PCR has the added potential advantages of providing Ureaplasma
species identification and same-day turnaround without the necessity of maintaining organism viability, culture is relatively simple and can often provide results in 24 to 48 h. Compared to traditional PCR, the new multiplex real-time PCR greatly reduced the false-negative rate in the detection of Ureaplasma
species as defined by culture positivity, making this assay even more attractive than traditional PCR in this setting.
Yi et al. (37
) developed a real-time PCR assay to simultaneously detect and discriminate between the two Ureaplasma
species in a single test with one pair of common primers for both species and two species-specific TaqMan probes. This assay, however, lacked analytical sensitivity, and its clinical sensitivity was lower than that of traditional PCR. In contrast, our multiplex real-time PCR found a total of six more U. parvum
strains alone or in combination than with our traditional PCR and one additional U. urealyticum
strain. No other published studies have provided a quantitative comparison of species differentiation using real-time PCR versus traditional PCR.
Molecular genotyping methods based on the mba
gene and its 5′ end have been explored as a replacement for the antibody-based phenotyping methods (5
) that have been in use for almost 3 decades since the original description of the 14-member serotyping scheme by Robertson and Stemke in 1982 (21
). However, using the limited sequence data available for previous genotyping methods, only partial serovar identification was possible. The four serovars of U. parvum
were readily differentiated from each other by traditional PCR with different sets of primers (13
) or by two multiplex real-time PCRs (5
). However, to distinguish the 10 serovars of U. urealyticum
is still challenging. Using single-stranded conformation polymorphism analysis, the 10 serovars were divided into two groups, A (serovars 2, 5, 8, and 9) and B (serovars 4, 7, 10, 11, 12, and 13) (18
). By PCR and sequencing, Kong et al. (14
) classified the 10 serovars into three subgroups, 1 (serovars 2, 5, 8, and 9), 2 (serovars 4, 10, 12, and 13), and 3 (serovars 7 and 11). The same investigators then provided better discrimination by dividing the 10 serovars into five MBA genotypes with the added individual separation of serovars 9 and 10, i.e., genotypes A (serovars 2, 5, and 8), B (serovar 10), C (serovars 4, 12, and 13), D (serovar 9), and E (serovars 7 and 11) (13
). This approach, using just the sequence of an mba
gene specific to each serovar, should be reconsidered in light of the whole genome sequences that show that all Ureaplasma
serovars encode multiple members of the phase-variable mba
In designing our serovar-specific real-time PCR assays, we sought to avoid the mba gene family because of its ubiquity within the ureaplasmas and because of its phase variability. This intent was thwarted by the high interserovar identity among the 14 serovars. The average difference among the 4 U. parvum genomes is 0.56%, and among the 10 U. urealyticum genomes, the average is 0.63%. While it is straightforward to design PCRs for the discrimination of any pair of serovars, the discrimination of 1 serovar from the other 13 is often very challenging. We sought to have all assays employ primers and probes; however, in many cases, this was not possible. We were even forced, in several cases, to target mba genes or mba paralogous gene family genes to obtain serovar identification. In those instances, we made sure our PCR targets did not span sites at which chromosome rearrangements took place during phase variation. Those sites could be identified by analyzing whole-genome shotgun sequencing assemblies. By computationally searching thousands of unique loci in each serovar and testing with 14 ATCC type stains, we created PCRs specific to all 14 serovars without any cross-reactions. Many PCRs predicted to be serovar specific failed when tested and were abandoned. Ultimately, each serovar primer/probe set we used contains at least one serovar-specific primer or probe. In the cases where the set could cross-react, leading to a significantly larger amplicon, the PCR conditions were adjusted to prevent it. The strategy to multiplex the 14 PCR assays, which should be more efficient, was not used because each PCR condition was unique and combination with another assay compromised the specificity and sensitivity of one or both assays (data not shown).
In conclusion, our new multiplex real-time PCR for the detection and discrimination of Ureaplasma species is robust and ready to replace the traditional PCR for clinical diagnostic purposes and is a suitable alternative to culture. We have also shown for the first time that all 14 serovar type strains of Ureaplasma spp. can be clearly differentiated from each other by using our novel real-time PCR technology, which overcomes many of the limitations that hampered the utilization of serologically based typing methods. This should greatly facilitate future investigations of ureaplasmas at the species and serovar levels under clinical conditions to assess differential pathogenicity. Our data regarding the monoplex serovar-specific PCR assays were limited to evaluations using individual type strains representing each serovar. We did not evaluate clinical specimens directly, nor have we applied this technology thus far to clinical isolates. The value of this assay as a diagnostic typing method may ultimately depend on the degree of genetic variability that exists among clinical isolates and the extent of horizontal gene transfer that may influence serovar designation. The analytical sensitivities we obtained suggest that our assay should meet the requirements for direct testing of clinical specimens that may contain serovars alone or in combination. Proof of this will require additional clinical studies, as will the evaluation of how well these assays perform with clinical isolates of unknown serovar status.