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High-resolution melt PCR analysis for genotyping of Ureaplasma parvum isolates directly from clinical samples
Matthew S Payne1, Tania Tabone, Matthew W Kemp
1School of Women's and Infants' Health, University of Western Australia, Subiaco, WA, Australia.
Abstract:
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.
Insights
A new high-resolution melt (HRM) PCR assay rapidly differentiates Ureaplasma parvum serovars directly from clinical samples. This cost-effective method improves diagnosis of Ureaplasma parvum infections in neonates and adults.
Area of Science:
- Microbiology
- Molecular Diagnostics
Background:
- Ureaplasma sp. infections are linked to various pathologies in neonates and adults.
- Ureaplasma parvum is the most prevalent human Ureaplasma species.
- Current serovar differentiation relies on gene sequencing, which can be time-consuming.
Purpose of the Study:
- To develop a rapid, single-step assay for differentiating the four Ureaplasma parvum serovars.
- To establish a cost-effective diagnostic tool for Ureaplasma parvum infections.
Main Methods:
- Development of a high-resolution melt (HRM) PCR assay using primers targeting conserved regions of the multiple-banded antigen (MBA) gene.
- Validation of the assay using known U. parvum strains and clinical swab samples.
- Comparison of HRM PCR with traditional sequencing methods.
Main Results:
- The HRM PCR assay accurately differentiated the four U. parvum serovars (1, 3, 6, and 14) with 100% accuracy on known strains.
- The assay successfully identified U. parvum serovars directly from clinical swabs.
- HRM PCR distinguished single and mixed serovar infections, though software limitations exist for precise mixed serovar composition identification.
- The assay did not amplify Ureaplasma urealyticum serovars.
Conclusions:
- HRM PCR offers a rapid, cost-effective, and sensitive method for differentiating U. parvum serovars.
- This assay can be directly applied to clinical samples, potentially reducing the need for microbial culture.
- The developed HRM PCR assay is a valuable tool for diagnosing and managing Ureaplasma parvum infections.
