Establishment of a method for single-cell level research on HBV using droplet digital PCR technology
MA Huajie1, WU Min1, ZHANG Zhanqing1, YUAN Zhenghong2, YI Zhigang1，2, ZHANG Xiaonan1，3
1. Shanghai Public Health Clinical Center, Fudan University, Shanghai 201508, China; 2. Key Laboratory of Medical Molecular Virology (MOE/NHC), School of Basic Medical Sciences, Shanghai Medical College, Fudan University, Shanghai 200032, China; 3. Faculty of Science and Technology, University of Canberra, Australia Capital Territory 2617, Australia
Abstract：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.
马华杰1，邬敏1，张占卿1，袁正宏2，易志刚1，2，张小楠1，3. 基于微滴数字PCR技术建立单细胞水平研究HBV的方法[J]. 微生物与感染, 2023, 18(5): 269-277.
MA Huajie1, WU Min1, ZHANG Zhanqing1, YUAN Zhenghong2, YI Zhigang1，2, ZHANG Xiaonan1，3. Establishment of a method for single-cell level research on HBV using droplet digital PCR technology. JOURNAL OF MICROBES AND INFECTIONS, 2023, 18(5): 269-277.
2023, Vol. 18 
