State Key Laboratory of Organ Failure Research, Guangdong Provincial Key Laboratory of Viral Hepatitis Research, Department of Infectious Diseases, Nanfang Hospital, Southern Medical University, Guangzhou, China.
Methods Mol Biol. 2024;2837:159-170. doi: 10.1007/978-1-0716-4027-2_14.
In recent years, serum hepatitis B virus (HBV) RNA has been identified as a promising noninvasive surrogate biomarker of intrahepatic covalently closed circular DNA (cccDNA), detection of which requires an invasive liver biopsy in patients with chronic HBV infection. It is impractical to detect intrahepatic cccDNA as a routine diagnosis for chronic hepatitis B (CHB) patients in clinical management. Here, we describe a detailed protocol for serum HBV RNA quantification, which can reflect the activity of intrahepatic cccDNA. The procedure includes three major steps: (1) Simultaneous isolation of HBV DNA and RNA from patients' serum, (2) DNase I digestion for removing HBV DNA contamination, and (3) HBV RNA quantification by one-step reverse transcription qPCR.
近年来，血清乙型肝炎病毒 (HBV) RNA 已被确定为一种很有前途的肝内共价闭合环状 DNA (cccDNA) 的非侵入性替代生物标志物，在慢性 HBV 感染患者中检测 cccDNA 需要进行侵入性肝活检。在临床管理中，将检测肝内 cccDNA 作为慢性乙型肝炎 (CHB) 患者的常规诊断是不切实际的。在这里，我们描述了一种详细的血清 HBV RNA 定量检测方案，该方案可以反映肝内 cccDNA 的活性。该程序包括三个主要步骤：（1）从患者血清中同时分离 HBV DNA 和 RNA，（2）用 DNA 酶 I 消化去除 HBV DNA 污染，以及（3）通过一步逆转录 qPCR 定量 HBV RNA。
