Detection and Characterization of Human Ureaplasma Species and Serovars by Real-time PCR
J Clin Microbiol. 2010 Jun 16; 48(8): 2715-23.
We designed primers and probes for detection and discrimination of Ureaplasma parvum and U. urealyticum and their 14 serovars by real-time PCR. Analytical sensitivity and specificity for 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 2.8x10(-2) CFU/mul PCR mixture for detecting U. parvum and 4.1x10(-2) CFU/mul PCR mixture for detecting U. urealyticum. Clinical specificity and sensitivity were proven by comparison with culture and traditional PCR. For detection of any Ureaplasma species, the clinical sensitivity and specificity of real-time PCR were 96.9% and 79.0%, respectively, using culture as 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 speciation PCR is specific and sensitive in 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.