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We describe the first use of pulsed field gel electrophoresis to genotype human Ureaplasma species. This technique can distinguish between U. urealyticum and U. parvum, differentiate most of the 14 serovars from one another, and identify differences among clinical isolates of the same serovar.

Prenatal intrauterine infection has been recognized as an important cause of premature birth, and Ureaplasma urealyticum is one of the commonest pathogens. U. urealyticum consists of 14 serovars that can be divided into two biovars (parvo and T960), and the pathogenicity of U. urealyticum may be different according to the biovar. To detect U. urealyticum and determine its biovar simultaneously, we developed a real-time polymerase chain reaction (PCR) assay targeting urease gene. The real-time PCR biovar-typed two reference strains and 42 culture isolates of U. urealyticum as correctly as conventional PCR with direct sequencing. Subsequently, 87 clinical specimens (amniotic fluid, cord blood, vaginal swab) were tested for culture, conventional PCR, and real-time PCR. When compared with conventional PCR, sensitivity and specificity of real-time PCR were 89.5 and 98.5%, respectively, and those of culture were 47.4 and 100%, respectively. Of 18 clinical specimens that were found positive and biovar-typed by real-time PCR, parvo biovar was 66.7% and T960 biovar was 33.3%. This real-time PCR assay can be useful for the simultaneous detection and biovar discrimination of U. urealyticum in clinical specimens. Further study to quantify U. urealyticum would be facilitated on the basis of this method.

Ureaplasma parvum and Ureaplasma urealyticum, also known as biovar parvum and biovar T960, respectively, could be associated with several disorders in men, women, and mainly, in newborn children with under weight. Several methods have been developed in order to identify the species or biovars of ureaplasmas. We developed a Multiplex-PCR method using the UPS-UPSA and UUS2-UUA2 primers, specific for U. parvum and U. urealyticum, respectively. This Multiplex-PCR method was used to identify cultures of clinical positive samples to Ureaplasma spp. by the "MYCOFAST Evolution-2" Kit. Of 56 positive cultures to Ureaplasma spp. from newborn children, 70% were U. parvum and 30% U. urealyticum; in 76 positive samples in women, 83% corresponded to U. parvum and 17% to U. urealyticum, while in 63 positive samples of men, 76% identified U. parvum and 24% U. urealyticum. The PCR-multiplex method showed specificity for the identification of the biovars or species of ureaplasmas of clinical interest.

Fourteen serotypes are currently recognized in the Ureaplasma urealyticum species. These serotypes have been divided into two genomic clusters or biovars by a large number of typing methods. The parvo-biovar includes strains of serotypes 1, 3, 6 and 14 and the T960-biovar, strains belonging to the ten other serotypes. In this study, arbitrarily primed polymerase chain reaction (AP-PCR) has been applied to the analysis of reference strains of the 14 U. urealyticum serotypes. By using two different sets of 10-mer oligonucleotide primers, the method allowed the clear differentiation between the two known biovars of the species. However, further differentiation within a same biovar was only achieved for a few standard strains of the T960-biovar analysed by using a pairwise combination of primers. The reproducibility of AP-PCR profiles was shown on strains tested after repeated subcultures and with different thermal cyclers. Additional experiments were performed on forty isolates of U. urealyticum recovered from subjects of various origins. They confirmed that AP-PCR was able to identify the strains at the biovar level. With reference to the other typing methods, AP-PCR is easy to perform and can be applied to large numbers of strains for epidemiological purposes.