Rex Wan-Hin Hui, Danny Ka-Ho Wong, Lung-Yi Mak, James Fung, Wai-Kay Seto, Man-Fung Yuen
Real-time polymerase chain reaction (PCR) is the current standard for serum HBV DNA measurement. However, conventional real-time PCR assays have technical limitations, and are not sensitive enough to detect low-level residual viremia in chronic hepatitis B (CHB) patients. We developed and validated a droplet digital PCR (ddPCR) assay for high-sensitivity detection of serum HBV DNA. A ddPCR assay was developed on the QX200 ddPCR System (Bio-Rad) for detection of serum HBV DNA in 200 μL of serum. The primers and probe were designed to target a highly-conserved region in the HBV X gene. The AcroMetrix HBV Panel (Thermo Fisher Scientific) and CHB patient samples were used for validation experiments to determine the assay sensitivity, specificity, linearity, intra-run variability, and inter-run variability. The ddPCR assay demonstrated lower limit of detection of 1.6 IU/mL and lower limit of quantification of 9.4 IU/mL for serum HBV DNA in probit regression. The assay also achieved excellent specificity (96.2%), linearity (R = 0.994, R2= 0.988, p < 0.001), intra-run variability (mean coefficient of variation [CV]: 0.69%, average intra-run difference: 0.026 log IU/mL), and inter-run variability (mean coefficient of variation [CV]: 4.54%, average inter-run difference: 0.18 log IU/mL). To conclude, we developed a robust ddPCR assay that achieved higher detection sensitivity with lower serum input volume than conventional real-time PCR assays. Our assay may be utilised for measuring residual viremia after nucleos(t)ide analogue therapy or for monitoring patients on novel HBV antivirals.
{"title":"Development and Validation of a High-Sensitivity Droplet Digital PCR Assay for Serum Hepatitis B Virus DNA Detection","authors":"Rex Wan-Hin Hui, Danny Ka-Ho Wong, Lung-Yi Mak, James Fung, Wai-Kay Seto, Man-Fung Yuen","doi":"10.1111/jvh.70023","DOIUrl":"https://doi.org/10.1111/jvh.70023","url":null,"abstract":"<p>Real-time polymerase chain reaction (PCR) is the current standard for serum HBV DNA measurement. However, conventional real-time PCR assays have technical limitations, and are not sensitive enough to detect low-level residual viremia in chronic hepatitis B (CHB) patients. We developed and validated a droplet digital PCR (ddPCR) assay for high-sensitivity detection of serum HBV DNA. A ddPCR assay was developed on the QX200 ddPCR System (Bio-Rad) for detection of serum HBV DNA in 200 μL of serum. The primers and probe were designed to target a highly-conserved region in the HBV X gene. The AcroMetrix HBV Panel (Thermo Fisher Scientific) and CHB patient samples were used for validation experiments to determine the assay sensitivity, specificity, linearity, intra-run variability, and inter-run variability. The ddPCR assay demonstrated lower limit of detection of 1.6 IU/mL and lower limit of quantification of 9.4 IU/mL for serum HBV DNA in probit regression. The assay also achieved excellent specificity (96.2%), linearity (<i>R</i> = 0.994, <i>R</i><sup>2</sup>= 0.988, <i>p</i> < 0.001), intra-run variability (mean coefficient of variation [CV]: 0.69%, average intra-run difference: 0.026 log IU/mL), and inter-run variability (mean coefficient of variation [CV]: 4.54%, average inter-run difference: 0.18 log IU/mL). To conclude, we developed a robust ddPCR assay that achieved higher detection sensitivity with lower serum input volume than conventional real-time PCR assays. Our assay may be utilised for measuring residual viremia after nucleos(t)ide analogue therapy or for monitoring patients on novel HBV antivirals.</p>","PeriodicalId":17762,"journal":{"name":"Journal of Viral Hepatitis","volume":"32 4","pages":""},"PeriodicalIF":2.5,"publicationDate":"2025-03-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1111/jvh.70023","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143629825","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}