在单细胞水平研究肝组织内乙型肝炎病毒(hepatitis B virus, HBV)是十分重要的，但相关研究技术尚处于推进阶段，不能满足临床需要。基于微滴数字聚合酶链反应技术(digital droplet polymerase chain reaction，ddPCR)，本研究建立了一套单细胞水平绝对定量HBV DNA和RNA的单细胞微滴数字PCR方法(single-cell ddPCR, sc-ddPCR)。通过混合不同比例的HBV阳性和阴性细胞来模拟HBV慢性感染状态下的肝脏，分析此方法检测HBV DNA的线性和检测下限。以cccDNA来源RNA(episome-derived RNA, eRNA)为例，进一步设计针对eRNA的sc-ddPCR检测方案。利用不同来源转录本在结构上的差异，设计针对eRNA的特异性引物探针体系，在HBV肝癌细胞系中验证此引物和探针体系的特异度，并利用梯度稀释的HBV细胞株分析此方法检测eRNA的线性和检测下限。结果显示，本研究建立了基于ddPCR的DNA和RNA的单细胞水平检测方法，该方法表现出良好的线性(HBV DNA：R²＝0.998 7；eRNA: R²＝0.942 5)，梯度稀释的HBV细胞株均能准确检测出阳性信号，具有高灵敏度(检测下限：HBV DNA为0.16%，eRNA为0.2%)。本研究建立的sc-ddPCR方法可针对多种DNA和RNA进行检测，为进一步研究肝组织内 HBV 病毒学活动提供了良好的技术支撑，可以更深入地分析肝组织内HBV的病毒学特征，从而有利于 HBV 治疗策略的优化和研发，具有广阔的应用前景。

The single-cell study of hepatitis B virus (HBV) in liver tissue is of paramount significance. However, the associated research technology is still in an advanced stage and represents an unmet clinical need. Based on ddPCR (digital droplet polymerase chain reaction), this study has pioneered a single-cell ddPCR (sc-ddPCR) method for the precise quantification of HBV DNA and RNA at the single-cell level. To ascertain the linearity and lower detection limit of HBV DNA, a series of experiments were conducted by simulating a chronic HBV infection in the liver through the mixing of various proportions of HBV-positive and negative cells. The study focused on the detection of episome-derived RNA (eRNA), developing a tailored protocol for its analysis. By capitalizing on the structural disparities in HBV transcripts, the study had formulated a specific primer and probe system for eRNA, the specificity of which was rigorously confirmed in HBV liver cancer cell lines. The linearity and lower detection limit of eRNA were scrutinized through the utilization of gradient-diluted standard cell lines. The results unequivocally demonstrated the establishment of an advanced single-cell detection method for HBV DNA and RNA based on ddPCR, characterized by exceptional linearity (HBV DNA: R²＝0.998 7; eRNA: R²＝0.942 5). Moreover, the method exhibited a remarkable sensitivity, as it consistently detected positive signals in gradient-diluted standard cell lines, with a lower detection limit of 0.16% for HBV DNA and 0.2% for eRNA. The sc-ddPCR method developed in this study is versatile, enabling simultaneous detection of DNA and RNA, and it serves as a robust technical foundation for further exploration of HBV virology activities within liver tissue. It is also invaluable for optimizing and advancing HBV treatment strategies, presenting promising prospects for broad applications.