Detection of Ureaplasma urealyticum by PCR and biovar determination by liquid hybridization

Abstract

An assay which combines the direct detection of Ureaplasma urealyticum with biovar determination was developed and applied to 618 urogenital specimens. U. urealyticum was detected by inhibitor-controlled PCR. A 429-bp fragment of the urease gene was amplified. The amplicons were labelled with digoxigenin during PCR. Biovar determination was performed by liquid hybridization with biotin-labelled biovar-specific probes, and the hybrids were detected with peroxidase-conjugated sheep anti-digoxigenin immunoglobulin G Fab fragments. Results of PCR and culture for 453 urogenital specimens from women and 105 urethral specimens from men could be compared. Among the specimens from women, 63% were PCR positive as well as culture positive, 0.9% were positive only by PCR, and 4% were positive only by culture. Among the specimens from men, 15% were PCR positive as well as culture positive, 1% were positive only by PCR, and 9% were positive only by culture. By using culture as the reference method, the PCR had a sensitivity of 94% and a specificity of 98% when applied to specimens from women and a sensitivity of 64% and a specificity of 99% when applied to specimens from men. Overall, 80% of the PCR-positive specimens contained biovar 1,13.5% contained biovar 2, and 6.5% contained both biovars.

FIG. 1

Electrophoretic analysis of amplicons obtained by PCR with species-specific primers U5 and U4. Lower band (429 bp), specific for U. urealyticum; higher band (657 bp), internal process control. Lane 1, 100-bp marker; lane 2, positive sample lacking the internal process control; lane 3, positive sample with the internal process control; lane 4, inhibitory sample; lane 5, negative sample; lane 6, negative sample (water); lane 7, 100-bp marker.

FIG. 2

Observed and estimated PCR-positive fraction as a function of the numbers of CCUs for the specimens.

FIG. 3

Alignment of the sequenced products of the 429-bp fragment of the urease gene. The sequences of serotype standard strains 1, 3, 6, and 14 (biovar 1) are compared to the published sequence of serotype standard strain 1 (13) designated Urease 1. The sequences of serotype standard strains 2, 4, 5, 7, 8, 9, 10, 11, and 13 (biovar 2) are compared to the published sequence of serotype standard strain 8 (2) designated Urease 2. The sequences of the probes that are not useful are marked by underlines, and the sequences of the useful probes are marked by double underlines.

FIG. 4

Competition during amplification of various ratios of biovar 1 DNA and biovar 2 DNA. Biovar 1 and biovar 2 DNAs were mixed at various ratios before amplification, but the total amount of DNA was kept constant.

FIG. 5

Liquid hybridization assay for assignment of U. urealyticum biovars, with the following results: hybridization with the biovar 1-specific probe, 1A to 3H and 4A to 4F; positive specimens containing biovar 1, 1A, 1B, 2C, 2F, and 2G; positive controls of biovar 1, 4A and 4B; positive controls of biovar 2, 4C and 4D; negative controls, 4E and 4F; hybridization with the biovar 2-specific probe, 5A to 7H and 8A to 8F. Positive specimens containing biovar 2, 6E, 7E, and 7G; positive controls of biovar 1, 8A and 8B; positive controls of biovar 2, 8C and 8D; negative controls, 8E and 8F.