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

Abstract

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.

In vitro activity of solithromycin and its metabolites, CEM-214 and N-acetyl-CEM-101, against 100 clinical Ureaplasma spp. isolates compared with azithromycin

“…Using real-time PCR, we previously confirmed that all of the UK isolates used in this study were U. parvum [23]. Subsequent analysis of the Australian isolates in the current study showed that 20% were U. urealyticum.…”

“…Payne et al [23] had already established species-level identification of UK triple-cloned isolates. For the Australian isolates, to determine whether the triple-cloned Ureaplasma spp.…”

Whole blood flow cytometric analysis of Ureaplasma-stimulated monocytes from pregnant women

“…A clinical isolate of UpSV3 was chosen for stimulation (Payne et al, 2014). Exponential phase cultures were quantified, as previously described (Beeton et al, 2012), and heat-killed by incubation of 2 mL cultures at 60 • C for 4 h. Cells collected by centrifugation at 15,000 × g, 4 • C for 20 min were resuspended to 10 7 CCU/mL in sterile 1× PBS (Sigma, St. Louis, MO, USA).…”

“…DNA extraction and PCR-HRM analysis of Ureaplasma spp. status was carried out as previously described (Payne et al, 2014).…”

One-step simultaneous detection ofUreaplasma parvumand genotypes SV1, SV3 and SV6 from clinical samples using PlexPCR technology

“…; Payne et al . ), to the best of our knowledge, none exist that are capable of concurrent genus and multiple genotype detection in a single reaction directly from clinical samples. Three years ago we developed a high‐resolution melt (HRM) PCR assay that was capable of detecting U. parvum and differentiating single genotype colonizations directly from clinical samples (Payne et al .…”