Background: First-void urine (FVU) provides a non-invasive method for collecting a wide range of biomarkers found in genital tract secretions. To optimize biomarker collection in FVU, this study investigated the impact of naturally present and supplemented precipitating agents: uromodulin (UMOD) and polyethylene glycol (PEG), on the concentration of human papillomavirus (HPV) pseudovirions (PsV), cell-free DNA (cfDNA), and cellular genomic DNA (gDNA) through centrifugation.
Methods: FVU samples from ten healthy female volunteers, along with a control sample, were spiked with seal herpesvirus 1 (PhHV-1) DNA, HPV16 plasmid DNA, and HPV16 PsV with an enhanced green fluorescent protein (EGFP) reporter. The samples were subjected to various concentration protocols involving PEG precipitation, low-speed centrifugation (5 min at 1000×g), and medium-speed centrifugation (1 h at 3000×g). Subsequently, quantitative PCR (qPCR) was used to assess cellular and cell-free glyceraldehyde-3-phosphate dehydrogenase (GAPDH) DNA, cell-free PhHV-1 and HPV16 DNA, and PsV (EGFP) DNA. In addition, UMOD levels were measured.
Results: The findings revealed that PEG significantly increased the concentration of cfDNA and gDNA in the pellet after centrifugation, with the most pronounced effect observed for cfDNA. Moreover, low-speed centrifugation without PEG effectively depleted cellular gDNA while preserving cfDNA in the supernatants. Pseudovirions were consistently pelleted, even with low-speed centrifugation, and a positive but not significant effect of PEG on PsV (EGFP) DNA yield in the pellet was observed. Additionally, a significant correlation was observed between UMOD and GAPDH, HPV16, and PsV (EGFP) DNA quantities in the pellet. Furthermore, large variations among the FVU samples were observed.
Conclusions: With this study, we provide novel insights into how various biomarker precipitation protocols, including both the properties of FVU and the use of PEG as a precipitating agent, influence the concentration of cfDNA, cellular gDNA, and pseudovirions.
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| http://www.ncbi.nlm.nih.gov/pmc/articles/PMC10873940 | PMC |
| http://dx.doi.org/10.1186/s40001-024-01719-5 | DOI Listing |
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