Comparison of two methods for extracting hepatitis B virus covalently closed circular DNA
HU Kongying, JIN Yutao, YE Jianyu, ZAI Wenjing, WANG Yang, LI Yumeng, CHEN Jieliang
Department of Medical Microbiology and Parasitology, MOE & NHC Key Laboratory of Medical Molecular Virology, School of Basic Medical Sciences, Fudan University, Shanghai 200032, China
Abstract:Hepatitis B virus (HBV) is a hepatotropic DNA virus that can deposit a covalently closed circular DNA form (cccDNA) in the nucleus of infected hepatocytes, which serves as a reservoir for HBV genome and the template for transcribing HBV RNAs. The persistence of cccDNA is the key of HBV chronicity and the main obstacle for HBV cure, and thus cccDNA has always been a focus of HBV research. The efficient extraction of cccDNA from cells is required for accurate detection and quantification. Hirt DNA extraction is a method for isolation of the extrachromosomal DNA in eukaryotes and has been applied for cccDNA extraction. However, the procedure of Hirt DNA extraction is laborious and time-consuming. To simplify the procedure, there has been reports of ”modified Hirt DNA extraction” combined with silicon-based resins column for rapid extraction of extrachromosomal DNA. However, the difference of extraction efficacy between the two methods remains unknown. Here, the two methods were parallelly compared by using Southern Blot and qPCR in various cell culture-based HBV transfection, replication and infection systems. The results suggested that “the modified Hirt-Silica Gel Membrane method” has the potential to enable a fast HBV cccDNA extraction with equally extraction efficacy and specificity compared with “the original Hirt-Phenol/Chloroform method”.
胡孔颖,金宇焘,叶建宇,宰文静,王洋,李雨檬,陈捷亮. 两种抽提乙型肝炎病毒共价闭合环状DNA方法的效果比较[J]. 微生物与感染, 2021, 16(5): 313-321.
HU Kongying, JIN Yutao, YE Jianyu, ZAI Wenjing, WANG Yang, LI Yumeng, CHEN Jieliang. Comparison of two methods for extracting hepatitis B virus covalently closed circular DNA. JOURNAL OF MICROBES AND INFECTIONS, 2021, 16(5): 313-321.