We demonstrated previously that urine contains low-molecular-weight (LMW) (<300 bp), circulation-derived DNA that can be used to detect cancer-specific mutations if a tumor is present. The goal of this study was to develop an assay to detect the colorectal cancer (CRC)-associated, circulation-derived, epigenetic DNA marker hypermethylated vimentin gene (mVIM) in the urine of patients with CRC. An artificial 18-nucleotide DNA sequence was tagged at the 5' end of the primers of the first PCR cycle to increase the amplicon size, which was then integrated into the primers of the second PCR cycle. A quantitative MethyLight PCR-based assay targeting a 39-nucleotide template was developed and used to quantify mVIM in CRC tissues and matched urine samples. mVIM was detected in 75% of LMW urine DNA samples from patients with CRC (n = 20) and in 10% of urine samples of control subjects with no known neoplasia (n = 20); 12 of 17 LMW urine DNA samples (71%) but only 2 of 17 high-molecular-weight urine DNA samples (12%) from patients with mVIM-positive tissues contained detectable mVIM, suggesting that the mVIM detected in LMW urine DNA is derived from the circulation. The detection of mVIM in urine was significantly associated with CRC compared with controls (P < 0.0001, by Fisher's exact test). A potential urine test for CRC screening using epigenetic markers is discussed.
Integrated hepatitis B virus (HBV) DNA, found in more than 85% of HBV-associated hepatocellular carcinomas (HBV-HCCs), can play a significant role in HBV-related liver disease progression. HBV-host junction sequences (HBV-JSs), created through integration events, have been used to determine HBV-HCC clonality. Here, we investigate the feasibility of analyzing HBV integration in a noninvasive urine liquid biopsy. Using an HBV-targeted next-generation sequencing (NGS) assay, we first identified HBV-JSs in eight HBV-HCC tissues and designed short-amplicon junction-specific polymerase chain reaction assays to detect HBV-JSs in matched urine. We detected and validated tissue-derived junctions in five of eight matched urine samples. Next, we screened 32 urine samples collected from 25 patients infected with HBV (5 with hepatitis, 10 with cirrhosis, 4 with HCC, and 6 post-HCC). Encouragingly, all 32 urine samples contained HBV-JSs detectable by HBV-targeted NGS. Of the 712 total HBV-JSs detected in urine, 351 were in gene-coding regions, 11 of which, including TERT (telomerase reverse transcriptase), had previously been reported as recurrent integration sites in HCC tissue and were found only in the urine patients with cirrhosis or HCC. The integration breakpoints of HBV DNA detected in urine were found predominantly (~70%) at a previously identified integration hotspot, HBV DR1-2 (down-regulator of transcription 1-2). Conclusion: HBV viral-host junction DNA can be detected in urine of patients infected with HBV. This study demonstrates the potential for a noninvasive urine liquid biopsy of integrated HBV DNA to monitor patients infected with HBV for HBV-associated liver diseases and the efficacy of antiviral therapy. (Hepatology Communications 2021;0:1-11).
Using PCR and alkaline gel electrophoresis, we show that, compared with DNA derived from virions used to establish infection, herpes simplex virus DNA derived from quiescently infected rat pheochromocytoma (PC12) cells in culture accumulates alkaline-labile lesions. That is, compared with equivalent amounts of virion DNA, viral DNA from nerve growth factor-differentiated longterm infected cells in culture is consistently 3 to 10 times more refractory to amplification by PCR. Despite using equal mole amounts of DNA isolated from quiescently infected cells (determined by quantitative Southern blots), DNA from quiescently infected cells could not be detected by PCR under conditions in which the virion-derived DNA was easily detected. Refractoriness to PCR was confirmed by analysis with a ligation-mediated PCR technique. The refractoriness was not the result of genomic circularization. The refractoriness was, however, related to the time that the quiescently infected cells had been maintained in culture. The refractoriness to PCR was taken as an indication that the viral DNA was damaged. This hypothesis was confirmed by showing that viral DNA from quiescently infected PC12 cells accumulated alkaline-labile DNA lesions, as determined by alkaline gel electrophoresis. The phenomenon was not limited to tissue culture, because viral DNA derived from the ganglia of latently infected mice is also 3 to 10 times more refractory to amplification than are equivalent amounts of virion-derived genomes. Taken together, these results represent the first evidence that herpes simplex virus DNA is physically damaged as a function of long-term infection. Implications for viral reactivation and pathogenesis are discussed.
Objective: To develop a new, rapid, and convenient technique for the diagnosis and typing of herpes simplex virus (HSV) in genital herpes (GH). Methods: Using samples from skin vesicle fluid and urogenital mucosal swabs of subjects with GH, conventional polymerase chain reaction (PCR) (directed to polymerase gene: PCRpG) were compared with a newly developed PCR (directed to HSV glycoprotein gene: PCRgG). Both PCR methods were compared with virus isolation culture (VI) with indirect immunofluorescent staining (IIF). Results: 80 samples from 40 GH patients (25 males) were tested. Positive results were seen in 52.5% (42/80) using PCRgG compared with 40% (32/80) by VI. Most of PCRgG positive samples (95.1%) were caused by HSV-2 infection. In samples from healing lesions, HSV was detected more often by PCRgG, than by VI. The results of typing by PCRgG and IIF were highly consistent. Conclusion: PCRgG is more sensitive than VI and PCRpG in detecting HSV in urogenital samples from subjects with GH. PCRgG is a convenient technique for the rapid detection and typing of GH. (Sex Transm Inf 1999;75:396-397)
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.