Development and Validation of a High-Sensitivity Droplet Digital PCR Assay for Serum Hepatitis B Virus DNA Detection

IF 2.5 3区 医学 Q2 GASTROENTEROLOGY & HEPATOLOGY Journal of Viral Hepatitis Pub Date : 2025-03-15 DOI:10.1111/jvh.70023
Rex Wan-Hin Hui, Danny Ka-Ho Wong, Lung-Yi Mak, James Fung, Wai-Kay Seto, Man-Fung Yuen
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Abstract

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.

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实时聚合酶链反应(PCR)是目前测量血清 HBV DNA 的标准。然而,传统的实时 PCR 检测方法存在技术局限性,其灵敏度不足以检测慢性乙型肝炎(CHB)患者的低水平残余病毒血症。我们开发并验证了一种用于高灵敏度检测血清 HBV DNA 的液滴数字 PCR(ddPCR)测定。我们在 QX200 ddPCR 系统(Bio-Rad)上开发了一种 ddPCR 检测方法,用于检测 200 μL 血清中的血清 HBV DNA。引物和探针是针对 HBV X 基因中的高保守区设计的。AcroMetrix HBV Panel(赛默飞世尔科技公司)和 CHB 患者样本被用于验证实验,以确定检测灵敏度、特异性、线性、运行内变异性和运行间变异性。在 probit 回归中,ddPCR 分析显示血清 HBV DNA 的检测下限为 1.6 IU/mL,定量下限为 9.4 IU/mL。该检测方法的特异性(96.2%)、线性度(R = 0.994,R2 = 0.988,p < 0.001)、运行内变异性(平均变异系数 [CV]:0.69%,运行中的平均差异为 0.026 log IU/m:0.026 log IU/mL)和运行间变异性(平均变异系数 [CV]:4.54%,平均运行间差异:0.026 log IU/mL):4.54%,平均运行间差异:0.18 log IU/mL0.18 log IU/mL)。总之,我们开发了一种稳健的 ddPCR 检测方法,与传统的实时 PCR 检测方法相比,它能以更低的血清输入量实现更高的检测灵敏度。我们的检测方法可用于测量核苷(t)ide 类似物治疗后的残余病毒血症,或用于监测使用新型 HBV 抗病毒药物的患者。
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来源期刊
Journal of Viral Hepatitis
Journal of Viral Hepatitis 医学-病毒学
CiteScore
6.00
自引率
8.00%
发文量
138
审稿时长
1.5 months
期刊介绍: The Journal of Viral Hepatitis publishes reviews, original work (full papers) and short, rapid communications in the area of viral hepatitis. It solicits these articles from epidemiologists, clinicians, pathologists, virologists and specialists in transfusion medicine working in the field, thereby bringing together in a single journal the important issues in this expanding speciality. The Journal of Viral Hepatitis is a monthly journal, publishing reviews, original work (full papers) and short rapid communications in the area of viral hepatitis. It brings together in a single journal important issues in this rapidly expanding speciality including articles from: virologists; epidemiologists; clinicians; pathologists; specialists in transfusion medicine.
期刊最新文献
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