Molecular genotyping of human Ureaplasma species based on multiple-banded antigen (MBA) gene sequences.

Kong, Fanrong; Ma, Zhiyong; James, Gregory; Gordon, Susanna; Gilbert, Gwendolyn L.

doi:10.1099/00207713-50-5-1921

Cited by 44 publications

(34 citation statements)

References 24 publications

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“…Irrespective of discrepancies in their findings, previously described assays for serovar characterization of U. parvum have been both laborious and expensive, combining the use of endpoint PCR and sequencing (11,29) or four separate single-plex (12) or two separate duplex (30) real-time PCR assays. Here, we have described the first assay for U. parvum serovar characterization using an HRM PCR approach.…”

Section: Discussionmentioning

confidence: 99%

High-Resolution Melt PCR Analysis for Genotyping of Ureaplasma parvum Isolates Directly from Clinical Samples

et al. 2014

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c Ureaplasma sp. infection in neonates and adults underlies a variety of disease pathologies. Of the two human Ureaplasma spp., Ureaplasma parvum is clinically the most common. We have developed a high-resolution melt (HRM) PCR assay for the differentiation of the four serovars of U. parvum in a single step. Currently U. parvum strains are separated into four serovars by sequencing the promoter and coding region of the multiple-banded antigen (MBA) gene. We designed primers to conserved sequences within this region for PCR amplification and HRM analysis to generate reproducible and distinct melt profiles that distinguish clonal representatives of serovars 1, 3, 6, and 14. Furthermore, our HRM PCR assay could classify DNA extracted from 74 known (MBA-sequenced) test strains with 100% accuracy. Importantly, HRM PCR was also able to identify U. parvum serovars directly from 16 clinical swabs. HRM PCR performed with DNA consisting of mixtures of combined known serovars yielded profiles that were easily distinguished from those for single-serovar controls. These profiles mirrored clinical samples that contained mixed serovars. Unfortunately, melt curve analysis software is not yet robust enough to identify the composition of mixed serovar samples, only that more than one serovar is present. HRM PCR provides a single-step, rapid, cost-effective means to differentiate the four serovars of U. parvum that did not amplify any of the known 10 serovars of Ureaplasma urealyticum tested in parallel. Choice of reaction reagents was found to be crucial to allow sufficient sensitivity to differentiate U. parvum serovars directly from clinical swabs rather than requiring cell enrichment using microbial culture techniques.

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Section: Discussionmentioning

confidence: 99%

High-Resolution Melt PCR Analysis for Genotyping of Ureaplasma parvum Isolates Directly from Clinical Samples

et al. 2014

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“…Molecular genotyping methods were more rapid and accurate, readily separating the two Ureaplasma species (9,27,35,43,56). However, due to limited sequence variation in the PCR targets, only partial separation of serovars was achieved (8,(20)(21)(22)(23). Recently, we developed a set of Ureaplasma speciesand serovar-specific real-time PCR assays that separate completely all 14 ATCC serovars type strains without cross-reactions (55).…”

mentioning

confidence: 99%

Extensive Horizontal Gene Transfer in Ureaplasmas from Humans Questions the Utility of Serotyping for Diagnostic Purposes

et al. 2011

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Ureaplasma parvum and Ureaplasma urealyticum are sexually transmitted, opportunistic pathogens of the human urogenital tract. There are 14 known serovars distributed between the two species. For decades, it has been postulated based upon limited data that virulence is related to serotype specificity. The results were often inconclusive due to the small sample size and extensive cross-reactivity between certain serovars. We developed real-time quantitative PCRs that allow reliable differentiation of the two species and type strains of each of the 14 serovars. To investigate species and serovar distributions, we typed 1,061 clinical isolates of human ureaplasmas from diverse patient populations. There was only a tenuous association between individual Ureaplasma serovars and certain patient populations. This may in part be explained by the fact that almost 40% of the isolates were genetic mosaics, apparently arising from the recombination of multiple serovars. This explains the extensive cross-reactivity based upon serotyping and the lack of consistent association of given serotypes with disease.

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“…However, using the limited sequence data available for previous genotyping methods, only partial serovar identification was possible. The four serovars of U. parvum were readily differentiated from each other by traditional PCR with different sets of primers (13) or by two multiplex real-time PCRs (5). However, to distinguish the 10 serovars of U. urealyticum is still challenging.…”

Section: Discussionmentioning

confidence: 99%

“…Molecular genotyping methods based on the mba gene and its 5Ј end have been explored as a replacement for the antibody-based phenotyping methods (5,(11)(12)(13)(14)(15)18) that have been in use for almost 3 decades since the original description of the 14-member serotyping scheme by Robertson and Stemke in 1982 (21). However, using the limited sequence data available for previous genotyping methods, only partial serovar identification was possible.…”

Section: Discussionmentioning

confidence: 99%

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

et al. 2010

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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.

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Molecular genotyping of human Ureaplasma species based on multiple-banded antigen (MBA) gene sequences.

Cited by 44 publications

References 24 publications

High-Resolution Melt PCR Analysis for Genotyping of Ureaplasma parvum Isolates Directly from Clinical Samples

High-Resolution Melt PCR Analysis for Genotyping of Ureaplasma parvum Isolates Directly from Clinical Samples

Extensive Horizontal Gene Transfer in Ureaplasmas from Humans Questions the Utility of Serotyping for Diagnostic Purposes

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

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