COMPARISON OF PCR, NESTED PCR, AND RANDOM AMPLIFIED POLYMORPHIC DNA PCR FOR DETECTION AND TYPING OF UREAPLASMA-UREALYTICUM IN SPECIMENS FROM PREGNANT-WOMEN

A pen assay, using three primer pairs, was developed for the detection of Ureaplasma urealyticum, parvo biovar; mba types 1, 3, and 6, in cu ltured clinical specimens. The primer pairs were designed by using the polymorphic base positions within a 310- to 311-bp fragment of the 5' end and upstream control region of the mba gene. The specificity of t he assay was confirmed with reference serovars 1, 3, 6, and 14 and by the amplified-fragment sizes (81 bp for mba 1, 262 bp for mba 3, and 1 93 bp for mba 6), A more sensitive nested PCR was also developed. This involved a first-step PCR, using the primers UMS-125 and UMA226, foll owed by the nested mba-type PCR described above, This nested PCR enabl ed the detection and typing of small numbers of U, urealyticum cells, including mixtures, directly in original clinical specimens. By using random amplified polymorphic DNA (RAPD) PCR with seven arbitrary prime rs, we were also able to differentiate the two biovars of U, urealytic um and to identify 13 RAPD-PCR subtypes, By applying these subtyping t echniques to clinical samples collected from pregnant women, we establ ished that (i) U. urealyticum is often a persistent colonizer of the l ower genital tract from early midtrimester until the third trimester o f pregnancy, (ii) mba type 6 was isolated significantly more often (P = 0.048) from women who delivered preterm than from women who delivere d at term, (iii) no particular ureaplasma subtype(s) was associated wi th placental infections and/or adverse pregnancy outcomes, and (iv) th e ureaplasma subtypes most frequently isolated from women were the sam e subtypes most often isolated from infected placentas.