共价闭合环状DNA（cccDNA）是乙肝病毒（HBV）持续感染和停药后复发的根本原因，目前尚无高灵敏、高特异的cccDNA检测方法。最新发现CRISPR技术有助于痕量核酸检测且灵敏性和特异性均较高；我们前期已成功验证Cas13a蛋白活性，筛选了cccDNA的特异性引物和crRNA序列等。基于此，提出“CRISPR联合滚环扩增（RCA）和PCR技术可高灵敏、高特异检测cccDNA”假说。本项目拟：①利用不降解质粒的ATP依赖的DNA酶（PSAD）消化松散环状DNA（rcDNA）和双链线性DNA；②通过RCA和PCR特异性扩增cccDNA；③设计crRNA特异识别cccDNA转录的单链RNA激活Cas13a，从而酶切带有荧光信号的报告RNA，建立cccDNA检测新方法；④利用临床样本对该方法进行评价和验证。本项目期望建立兼具高灵敏和高特异的cccDNA检测新方法，为指导HBV治疗提供技术支撑。

The hepatitis B virus (HBV) is a global public health problem, with more than 240 million chronically infected individuals worldwide. HBV has a small (3.2 kb), partially double-stranded genome and replicates through reverse transcription of pregenomic RNA. The covalently closed circular DNA (cccDNA) in the nucleus, which serves as the template for transcription of pgRNA and other subgenomic RNAs, is initially formed immediately after HBV entry and persists inside the host cells. cccDNA has been widely recognized as the molecular basis of HBV persistence and, as such, also represents a target for next-generation therapeutics...Elimination of cccDNA is the ultimate goal of curative therapies for HBV. There is a clear need for specific assays that can detect HBV cccDNA in presence do viral replicative intermediate DNA including relaxed circular DNA(rcDNA). Southern blotting can effectively distinguish cccDNA and rcDNA based on differences in electrophoretic mobility, but the procedure is labor intensive. Quantitative PCR(qPCR) procedures that amplify across the gaps present in rcDNA can also achieve some specificity for cccDNA, but it is unclear whether the specificity is sufficient to guarantee that other rcDNA is not also being detected. Digital PCR is an effective method for the detection of HBV cccDNA. It has good sensitivity and specificity, but it is expensive, and requires special equipment, therefore digital PCR cannot be widely used. Therefore, the construction of a new method for detecting HBV cccDNA with high sensitivity and specificity needs further research...Clustered regularly interspaced short palindromic repeats (CRISPR) are a family of DNA sequences found within the genomes of prokaryotic organisms. CRISPR-associated (Cas) immune system has been applied in molecular biology to target and cleave specific nucleic acid sequences, which is commonly used in gene editing. Furthermore, upon binding to target double-stranded DNA (dsDNA) or RNA, several Cas proteins can be activated and unleash the nonspecific endoribonuclease activity to degrade the single--stranded DNA (ssDNA) and RNA and thus provide a novel diagnostic approach for nuclei acid detection. A recent articles on CRISPR technology to establish nucleic acid detection methods has been published in Science and Nature, and proved that CRISPP-Cas biology promises rapid, accurate,and portable diagnostic tools and will facilitate early disease detection and intervention. Therefore, we propose a hypothesis: CRISPR-Cas technology combined with rolling circle amplification (RCA) and PCR technology can highly sensitive and specific detect of HBV cccDNA...To verify this hypothesis, we will do as below ① to digest loose circle rcDNA and double-stranded linear DNA using PSAD enzymes; ② to amplify specific HBV cccDNA fragments by RCA and PCR; ③ to transcribe HBV cccDNA into single-stranded RNA, and use CRISPR crRNA to specifically bind the target gene fragment; ④ to establish a new method for detecting HBV cccDNA by using Cas13a digestion activity to digest a reporter promoter with a fluorescent signal; ⑤ to further certificate the clinical application of new method using patient specimens. ..In summary, through this study, we will establish a new method for detecting cccDNA with high sensitivity and specificity, which will provide a solid foundation for understanding the pathogenic mechanism of HBV and evaluating the antiviral efficacy.