Impact of Collection Volume and DNA Extraction Method on the Detection of Biomarkers and HPV DNA in First-Void Urine

Abstract

The potential of first-void (FV) urine as a non-invasive liquid biopsy for detection of human papillomavirus (HPV) DNA and other biomarkers has been increasingly recognized over the past decade. In this study, we investigated whether the volume of this initial urine stream has an impact on the analytical performance of biomarkers. In parallel, we evaluated different DNA extraction protocols and introduced an internal control in the urine preservative. Twenty-five women, diagnosed with high-risk HPV, provided three home-collected FV urine samples using three FV urine collection devices (Colli-Pee) with collector tubes that differ in volume (4, 10, 20 mL). Each collector tube was prefilled with Urine Conservation Medium spiked with phocine herpesvirus 1 (PhHV-1) DNA as internal control. Five different DNA extraction protocols were compared, followed by PCR for GAPDH and PhHV-1 (qPCR), HPV DNA, and HBB (HPV-Risk Assay), and ACTB (methylation-specific qPCR). Results showed limited effects of collection volume on human and HPV DNA endpoints. In contrast, significant variations in yield for human endpoints were observed for different DNA extraction methods (p < 0.05). Additionally, the potential of PhHV-1 as internal control to monitor FV urine collection, storage, and processing was demonstrated.

Rapid Detection of Antimicrobial Resistance in Mycoplasma genitalium by High-Resolution Melting Analysis with Unlabeled Probes

“…The human β-globin ( HBB ) and mgpa allele were used as internal quality controls to ensure the performance of nucleic acid extraction and species identification, respectively. The HBB gene is widely recognized as an internal control gene in human samples that often coexists with target genes in clinical samples ( 41 , 42 ). Because there are only slight differences in the T m value (<0.3°C) between mutations S83N and D87N in the combined products, using assay 2, it is difficult to quickly distinguish between these mutations on samples with poor quality (such as in cases with redundant salt ions and proteins in the sample).…”

Clinical performance of a newly developed domestic urine‐based HPV test for cervical cancer screening in China

“…GenPlex ® HPV genotyping assay could realize many sample-in-result-out multiplexed molecular detection. In one test, 24 HPV genotypes can be detected and genotyped simultaneously (HPV 6,11,16,18,31,33,35,39,42,43,44,…”

“…Currently, most studies determined the potential of available HPV tests applied in urine which had been developed for cervical samples 12–14 . As expected, a discrepancy in the findings has been observed, indicating a lack of standardized HPV tests for urine 15,16 . Considering the lower viral load in urine compared with the cervical exfoliated sample, real‐time polymerase chain reaction (PCR) based HPV tests could improve the performance of HPV detection in urine and reduce variability than other ones 17 .…”

“…[12][13][14] As expected, a discrepancy in the findings has been observed, indicating a lack of standardized HPV tests for urine. 15,16 Considering the lower viral load in urine compared with the cervical exfoliated sample, real-time polymerase chain reaction (PCR) based HPV tests could improve the performance of HPV detection in urine and reduce variability than other ones. 17 Thus, it was necessary to develop a urine-based HPV test.…”

Dynamics of methylated cell-free DNA in the urine of non-small cell lung cancer patients

“…The urinary cfDNA was extracted from 20 mL (patients) or 40 mL (controls) urine supernatant using the Quick DNA urine kit (Zymo Research, Irvine, CA, US). Previous research showed that differences in urine collection volume in a similar range (4-20 mL) have limited effects on DNA yield, eliminating this potential bias [26]. DNA concentration was measured using the Qubit™ dsDNA HS Assay (Invitrogen, Carlsbad, CA, US).…”