Detection and Characterization of Human Ureaplasma Species and Serovars by Real-Time PCR

Abstract

We designed primers and probes for the detection and discrimination of Ureaplasma parvum and U. urealyticum and their 14 serovars by real-time PCR. The analytical sensitivity and specificity of the multiplex species-specific PCR were determined by testing corresponding American Type Culture Collection (ATCC) type strains, 47 other microbial species, and human genomic DNA. The limits of the multiplex PCR were 2.8 ؋ 10 ؊2 CFU/l PCR mixture for detecting U. parvum and 4.1 ؋ 10 ؊2 CFU/l PCR mixture for detecting U. urealyticum. Clinical specificity and sensitivity were proven by comparison with culture and traditional PCR. For the detection of any Ureaplasma species, the clinical sensitivity and specificity of real-time PCR were 96.9% and 79.0%, respectively, using culture as a reference. Multiplex real-time PCR was also more sensitive than traditional PCR in discriminating the two Ureaplasma species in culture-positive subcultures. Each of the 14 monoplex serovar-specific PCR assays was specific for the corresponding ATCC type strain serovar. This new species identification PCR is specific and sensitive in the detection of Ureaplasma species in clinical specimens, and the serovar-specific PCR assays are the first set of complete genotypic assays to differentiate all 14 known Ureaplasma serovars. These assays provide quick and reliable means for investigating the epidemiology and pathogenicity of ureaplasmas at the serovar level.

Ureaplasma urealyticum and Ureaplasma parvum in women of reproductive age

“…Zdorowska-Stefanov et al [16] found slightly lower ureaplasma prevalence (29.8% of 541 women tested), while Kechagia et al [17] found slightly higher prevalence (37.0% of 369 women tested). Molecular based studies have shown that most of ureaplasma isolates thus far considered UU were actually UP [18][19][20]. According to Kacerovský et al [21], the prevalence of UP in a cohort of healthy women who were not pregnant was 57%, which is a much higher prevalence than those of other genital mycoplasmas, viruses, Chlamydia or GBS infections.…”

Detection of Potential Sexually Transmitted Bacterial Pathogens by Molecular Methods

“…To characterize the ureaplasma species at the serovar level by PCR, genotyping primers based on the MBA gene and its 5′end upstream regions have been designed previously and partial serovar identification was achieved. In combination with direct sequencing or restriction enzyme analysis, these assays were capable of distinguishing ureaplasma serovars, and subtypes within serovars [19].…”

“…PCR-based methods are also becoming an important alternative to conventional culture for initial detection of ureaplasmas in clinical specimens and have the additional advantage of discrimination between the two Ureaplasma species [19]. Classic microbiological culture techniques are much less sensitive than PCR; less than half of the PCR positive probes in semen, urine, prostatic secretion, cervical swab, amniotic fluid, and vaginal specimen showed a positive culture [15,20].…”

“…Real-time TaqMan PCR assays have been developed that allow rapid, specific, sensitive, and quantitative detection with a 100 times greater sensitivity than conventional PCR, using the same primer sets and cycling conditions [19,21]. Convenient differentiation of U. parvum and U. urealyticum is also possible with real-time TaqMan PCR or traditional PCR assays; it can also be used to discriminate among all of the serovars [1,21].…”

“…Sophisticated nucleic acid amplification tests are necessary to discriminate between the two Ureaplasma spp, this is the reason of the lack of species determination in most studies until the past few years [21]. Gene targets for PCR assays used to detect ureaplasmas and to define species and subtypes have included the subunits of urease gene, 16S rRNA genes and the multiple-banded antigen gene (MBA) [19][20][21]. To characterize the ureaplasma species at the serovar level by PCR, genotyping primers based on the MBA gene and its 5′end upstream regions have been designed previously and partial serovar identification was achieved.…”