切换至 "中华医学电子期刊资源库"
高级检索
中华实验和临床感染病杂志(电子版)

中华实验和临床感染病杂志(电子版) ›› 2022, Vol. 16 ›› Issue (05) : 320 -327. doi: 10.3877/cma.j.issn.1674-1358.2022.05.006

论著

基于规律成簇的间隔短回文重复序列及其相关蛋白技术检测乙型肝炎病毒共价闭合环状DNA方法的建立
王俊文1, 田原2, 范子豪2, 徐玲2, 高耀2, 曹亚玲2, 潘桢桢2, 张向颖2, 宋岩1, 任锋2,†()   
  1. 1. 100022　北京，北京市垂杨柳医院检验科
    2. 100069　北京，首都医科大学附属北京佑安医院/北京肝病研究所
  • 收稿日期:2022-06-24 出版日期:2022-10-15
  • 通信作者: 任锋
  • 基金资助:
    国家自然科学基金项目(No. 81770611、82002243); 北京自然科学基金和北京市教委联合资助重点项目(No. KZ202010025035); 首都卫生发展科研专项重点攻关项目(No.首发2020-1-1151、首发2021-1G-2181); 北京市科技计划"首都临床诊疗技术研究及示范应用"专项课题(No. Z191100006619096、Z191100006619097); 北京市优秀人才培养项目(No. 2018000021469G289); 北京市医院管理中心"青苗"计划专项经费资助(No. QML20201702)

Establishment of a method for the detection of hepatitis B virus covalently closed circular DNA based on clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated proteins technology

Junwen Wang1, Yuan Tian2, Zihao Fan2, Ling Xu2, Yao Gao2, Yaling Cao2, Zhenzhen Pan2, Xiangying Zhang2, Yan Song1, Feng Ren2,†()   

  1. 1. Department of Clinical Laboratory, Beijing Chuiyangliu Hospital, Beijing 100022, China
    2. Beijing Institute of Hepatology, Beijing Youan Hospital, Capital Medical University, Beijing 100069, China
  • Received:2022-06-24 Published:2022-10-15
  • Corresponding author: Feng Ren
全文 ( ) 下载PDF ( ) 导出引用
引用本文：

王俊文, 田原, 范子豪, 徐玲, 高耀, 曹亚玲, 潘桢桢, 张向颖, 宋岩, 任锋. 基于规律成簇的间隔短回文重复序列及其相关蛋白技术检测乙型肝炎病毒共价闭合环状DNA方法的建立[J]. 中华实验和临床感染病杂志(电子版), 2022, 16(05): 320-327.

Junwen Wang, Yuan Tian, Zihao Fan, Ling Xu, Yao Gao, Yaling Cao, Zhenzhen Pan, Xiangying Zhang, Yan Song, Feng Ren. Establishment of a method for the detection of hepatitis B virus covalently closed circular DNA based on clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated proteins technology[J]. Chinese Journal of Experimental and Clinical Infectious Diseases(Electronic Edition), 2022, 16(05): 320-327.

目的

建立一种基于规律成簇的间隔短回文重复序列及其相关蛋白（CRISPR/Cas13a）的乙型肝炎病毒（HBV）共价闭合环状DNA（HBV cccDNA）检测方法。

方法

提取2017年6月至2020年10月于首都医科大学附属北京佑安医院就诊的4例乙型肝炎患者肝脏总DNA后，使用Hind Ⅲ内切酶和质粒安全性ATP依赖DNA酶（PSAD）分别进行酶切；根据松弛环状DNA（rcDNA）和cccDNA的结构差异，设计特异性扩增HBV cccDNA的引物，对酶切后的产物进行滚环扩增（RCA）和PCR扩增；并筛选crRNA，建立基于CRISPR/Cas13a技术的HBV cccDNA检测新方法。

结果

利用α-1-抗胰蛋白酶（A1AT）和HBV表面抗原（HBsAg）引物对双重酶切后的产物进行扩增，验证产物中HBV基因组的存在；利用HBV cccDNA和HBV rcDNA引物对PSDA酶切后的产物扩增，验证了产物中只存在HBV cccDNA；利用RCA后的阳性样本作为模板梯度稀释，然后进行PCR扩增转录后使用CRISPR/Cas13a检测，计算出检测下限为10拷贝/μl。

结论

本研究建立了RCA-PCR-CRISPR-Cas13a的新型检测方法，可对HBV cccDNA进行高灵敏度和高特异性检测，为乙型肝炎患者抗病毒治疗评估、治疗终点的确定以及调整治疗方案提供了有效的监测手段。

关键词: 肝炎病毒，乙型, 共价闭合环状DNA, 规律成簇的间隔短回文重复序列及其相关蛋白, 微滴数字聚合酶链反应, 荧光定量聚合酶链反应
Objective

To establish a clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated proteins (CRISPR/Cas13a)-based detection method for hepatitis B virus covalently closed circular DNA (HBV cccDNA).

Methods

After extracting the total DNA from the livers of 4 patients with hepatitis B collected in Beijing You’an Hospital, Capital Medical University from June 2017 to October 2020, total DNA was digested with Hind Ⅲ endonuclease and plasmid-safe ATP-dependent DNase (PSAD), respectively; According to the structural differences between relaxed circular DNA (rcDNA) and cccDNA, primers for specific amplification of HBV cccDNA were designed, and the products after digestion were subjected to rolling circle amplification (RCA) and PCR amplification; And crRNA was screened to establish a new method for HBV cccDNA detection based on CRISPR/Cas13a technology.

Results

Alpha-1 antitrypsin (A1AT) and hepatitis B virus surface antigen (HBsAg) primers were used to amplify the double digested product to verify the existence of hepatitis B virus genome in the product; Using HBV cccDNA and HBV rcDNA primers to amplify the product after PSDA digestion, it was verified that only HBV cccDNA exists in the product. The positive sample after RCA was used as a template for gradient dilution, and then PCR amplification was performed and CRISPR/Cas13a detection was used after transcription. The lower limit of detection was calculated to be 10 copies/μl.

Conclusions

A novel detection method of RCA-PCR-CRISPR-Cas13a was established, which can detect HBV cccDNA with high sensitivity and high specificity, and provide an effective monitoring method for the evaluation of antiviral therapy of hepatitis B patients, the determination of treatment endpoints, and the adjustment of treatment plans.

Key words: Hepatitis B virus, Covalently closed circular, deoxyribonucleic acid, Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated proteins, Droplet digital polymerase chain reaction, Real-time quantitative PCR
表1 引物及探针序列
名称 序列（5'→3'）
HBV cccDNA-F GGGGCGCACCTCTCTTTA
HBV cccDNA-R AGGCACAGCTTGGAGGC
HBV cccDNA-probe TCACCTCTGCCTAATCATCTC
β-actin-F ACTGTGCCCATCTACGAGG
β-actin-R CAGGCAGCTCGTAGCTCTT
β-actin-probe CGGGAAATCGTGCGTGAC
HBV rcDNA-F GTTGCCCGTTTGTCCTCTAATTC
HBV rcDNA-R GGAGGGATACATAGAGGTTCCTTGA
A1AT-F TTCCCTGGTCTGAATGTGTG
A1AT-R ACTGTCCCAGGTCAGTGGTG
HBsAg-F TCACAATACCGCAGAGTC
HBsAg-R ACATCCAGCGATAACCAG
R1 ACCTATTCTCCTCCC
R2 ATGCAACTTTTTCAC
R3 GGCCCACATATTGT
R4 AATCCTCACAATACC
R5 CTAGCAGAGCTTGGT
R6 CCTTTGTCCAAGGGC
R7 TAGAAGAAGAACTCC
R8 CCTATGGGAGTGGGC
表2 crRNA相关引物序列
名称 序列（5'→3'）
T7-crRNA-F TAATACGACTCACTATAGGGGATTTAGACTACCCCAA
HBV-1-crRNA GGGGATTTAGACTACCCCAAAAACGAAGGGGACTAAAAC TCACCTCTGCCTAATCATCTCTTGTTCA
HBV-1R TGAACAAGAGATGATTAGGC
HBV-2-crRNA GGGGATTTAGACTACCCCAAAAACGAAGGGGACTAAAACAACTTTTTCACCTCTGCCTAATCATCTC
HBV-2R GAGATGATTAGGCAGAGGTG
HBV-3-crRNA GGGGATTTAGACTACCCCAAAAACGAAGGGGACTAAAAC TTTTCACCTCTGCCTAATCATCTCTTGT
HBV-3R ACAAGAGATGATTAGGCAGA
图1 RCA-PCR-CRISPR-Cas13a检测示意图
图2 肝组织总DNA、Hind Ⅲ内切酶和PSAD酶切后的电泳图注：A：4个肝组织样本的总DNA条带亮度均较明显；B：Hind Ⅲ酶切后的条带亮度基本保持不变；C：PSAD酶切后已不见条带。每个样本进行3次重复实验
图3 Southern blot检测双重酶切后的产物注：A：地高辛标记HBV cccDNA探针检测结果；B：双重酶切后的产物检测结果，每个样本做2个重复实验
图4 A1AT和HBsAg引物扩增双重酶切后产物的电泳图注：A：A1AT引物扩增双重酶切后的产物，未出现目标条带；B：HBsAg引物扩增双重酶切后的产物，出现4个较明显的目标条带。每个样本进行3次重复实验
图5 双重酶切后HBV cccDNA引物扩增的电泳图注：HBV cccDNA引物扩增双重酶切后的产物，出现了较明显的目标条带。每个样本进行3次重复实验
图6 数字PCR检测RCA扩增前后的微滴散点图注：A：数字PCR检测RCA扩增前产物的阳性微滴数量较少；B：数字PCR检测RCA扩增后产物的阳性微滴数量显著增加。进行3次重复检测
图7 crRNA的筛选注：在检测60 min时，crRNA3的荧光值最高。每个crRNA检测进行3次重复
图8 CRISPR-cas13a系统的检测注：A：CRISPR-Cas13a系统检测梯度稀释的HBV cccDNA样本；B：A图在30 min时扣除背景的荧光值。每个梯度的浓度进行3次重复检测（*：P < 0.05、**：P < 0.01、***：P < 0.001）
[1]
Razavi-Shearer D, Gamkrelidze I, Nguyen MH, et al. Global prevalence, treatment, and prevention of hepatitis B virus infection in 2016: a modelling study[J]. Lancet Gastroenterol Hepatol,2018,3(6):383-403.
[2]
Revill PA, Chisari FV, Block JM, et al. A global scientific strategy to cure hepatitis B[J]. Lancet Gastroenterol Hepatol,2019,4(7):545-558.
[3]
Fung S, Choi H, Gehring A, et al. Getting to HBV cure: The promising paths forward[J]. Hepatology,2022,76(1):233-250.
[4]
Werle-Lapostolle B, Bowden S, Locarnini S, et al. Persistence of cccDNA during the natural history of chronic hepatitis B and decline during adefovir dipivoxil therapy[J]. Gastroenterology,2004,126(7): 1750-1758.
[5]
Lai CL, Wong D, Ip P, et al. Reduction of covalently closed circular DNA with long-term nucleos(t)ide analogue treatment in chronic hepatitis B[J]. J Hepatol,2017,66(2):275-281.
[6]
Wong G, Gane E, Lok A. How to achieve functional cure of HBV: Stopping NUCs, adding interferon or new drug development?[J]. J Hepatol,2022,76(6):1249-1262.
[7]
Gootenberg JS, Abudayyeh OO, Lee JW, et al. Nucleic acid detection with CRISPR-Cas13a/C2c2[J]. Science,2017,356(6336):438-442.
[8]
Chen JS, Ma E, Harrington LB, et al. CRISPR-Cas12a target binding unleashes indiscriminate single-stranded DNase activity[J]. Science, 2018,360(6387):436-439.
[9]
Myhrvold C, Freije CA, Gootenberg JS, et al. Field-deployable viral diagnostics using CRISPR-Cas13[J]. Science,2018,360(6387):444-448.
[10]
Kaminski MM, Abudayyeh OO, Gootenberg JS, et al. CRISPR-based diagnostics[J]. Nat Biomed Eng,2021,5(7):643-656.
[11]
李忠斌. 慢性乙型肝炎耐药患者外周血单个核细胞HVB cccDNA耐药相关基因突变特点研究[D]. 中国人民解放军医学院, 2012.
[12]
Kellner MJ, Koob JG, Gootenberg JS, et al. SHERLOCK: nucleic acid detection with CRISPR nucleases[J]. Nat Protoc,2019,14(10):2986-3012.
[13]
Lucifora J, Protzer U. Attacking hepatitis B virus cccDNA--The holy grail to hepatitis B cure[J]. J Hepatol,2016,64(Suppl 1):S41-S48.
[14]
Tsai KN, Kuo CF, Ou JJ. Mechanisms of hepatitis B virus persistence[J]. Trends Microbiol,2018,26(1):33-42.
[15]
Hoofnagle JH. Reactivation of hepatitis B[J]. Hepatology,2009,49 (Suppl5):S156-S165.
[16]
潘玉，王莉娜，宋正霞, 等. 恩替卡韦与干扰素治疗HBeAg阳性慢性乙型肝炎的疗效[J/CD]. 中华实验和临床感染病杂志(电子版),2016,10(4):392-395.
[17]
田原，徐玲，范子豪, 等. 基于微滴数字PCR技术建立HBV共价闭合环状DNA的检测方法[J]. 临床肝胆病杂志,2021,37(8):1806-1810.
[18]
Xu CH, Li ZS, Dai JY, et al. Nested real-time quantitative polymerase chain reaction assay for detection of hepatitis B virus covalently closed circular DNA[J]. Chin Med J (Engl),2011,124(10):1513-1516.
[19]
Caviglia GP, Abate ML, Tandoi F, et al. Quantitation of HBV cccDNA in anti-HBc-positive liver donors by droplet digital PCR: A new tool to detect occult infection[J]. J Hepatol,2018,69(2):301-307.
[20]
Li X, Zhao J, Yuan Q, et al. Detection of HBV covalently closed circular DNA[J]. Viruses,2017,9(6):139.
[21]
Zhang H, Tu T. Approaches to quantifying hepatitis B virus covalently closed circular DNA[J]. Clin Mol Hepatol,2022,28(2):135-149.
[22]
Abudayyeh OO, Gootenberg JS, Konermann S, et al. C2c2 is a single-component programmable RNA-guided RNA-targeting CRISPR effector[J]. Science,2016,353(6299):f5573.
[23]
Gootenberg JS, Abudayyeh OO, Kellner MJ, et al. Multiplexed and portable nucleic acid detection platform with Cas13, Cas12a, and Csm6[J]. Science,2018,360(6387):439-444.
[24]
Qin P, Park M, Alfson K J, et al. Rapid and fully microfluidic Ebola virus detection with CRISPR-Cas13a[J]. ACS Sens,2019,4(4):1048-1054.
[25]
Liu Y, Xu H, Liu C, et al. CRISPR-Cas13a nanomachine based simple technology for avian influenza A (H7N9) virus on-site detection[J]. J Biomed Nanotechnol,2019,15(4):790-798.
[1] 葛飞霞, 蒋银, 杨丹. 酶联免疫吸附测定法与PCR仪检测在乙型肝炎诊断中的临床应用[J]. 中华危重症医学杂志(电子版), 2023, 16(04): 310-315.
[2] 马娟, 唐仕芳, 刘慧敏, 张娅琴, 邓义娟, 汪丽, 李力. 《乙型肝炎病毒母婴传播预防临床指南(2020)》更新要点解读[J]. 中华妇幼临床医学杂志(电子版), 2021, 17(05): 516-526.
[3] 王迎迎, 谢平. 乙型肝炎病毒感染合并肺结核患者发生肝损伤的危险因素及预测模型构建[J]. 中华实验和临床感染病杂志(电子版), 2023, 17(04): 267-273.
[4] 庄虔莹, 李丽, 王文静, 康晓迪, 王素萍. 乙型肝炎病毒对体外受精-胚胎移植患者妊娠结局的影响[J]. 中华实验和临床感染病杂志(电子版), 2022, 16(06): 426-430.
[5] 李传举, 刘林月, 王美, 李昕, 韩祥辉, 贾海永. 乙型肝炎病毒感染模型研究进展[J]. 中华实验和临床感染病杂志(电子版), 2022, 16(06): 361-365.
[6] 曹秀贞, 易为, 王夫川, 杨秀梅. 乙型肝炎病毒感染孕妇替比夫定母婴阻断及其对婴儿乙肝疫苗免疫应答的影响[J]. 中华实验和临床感染病杂志(电子版), 2022, 16(03): 158-164.
[7] 纪世博, 庄立伟, 张雨, 李贲, 程丹颖, 刘顺爱, 成军, 邢卉春. 巨噬细胞游走抑制因子在乙型肝炎病毒相关性肝细胞癌中的调控作用[J]. 中华实验和临床感染病杂志(电子版), 2021, 15(06): 379-384.
[8] 王彩英, 何明, 刘玉环, 何树新, 庞琳. 慢性乙型肝炎病毒感染母亲母乳喂养婴儿乙型肝炎病毒表面抗体水平监测及临床意义[J]. 中华实验和临床感染病杂志(电子版), 2021, 15(05): 344-349.
[9] 刘蜜, 向田, 李叶静, 朱焦. 血液miRNA-548ah在慢性乙型肝炎病毒感染不同时期的表达及其临床价值[J]. 中华实验和临床感染病杂志(电子版), 2021, 15(05): 337-343.
[10] 张荣, 唐雍艳, 喻茜, 丁志云, 朱莉, 姚杰, 赵继, 张玉, 马德明, 张丽娜, 姚小英, 丁善文, 石尚虎, 贾玉芳, 沈静, 王海燕, 龚婵聪, 沈秀娟, 李明, 钱峰, 朱传武. 个体化方案阻断乙型肝炎病毒母婴传播十年数据分析[J]. 中华实验和临床感染病杂志(电子版), 2021, 15(05): 303-310.
[11] 覃亚勤, 秦英梅. 8 369例人类免疫缺陷病毒感染/获得性免疫缺陷综合征患者合并乙型肝炎病毒和（或）丙型肝炎病毒感染临床分析[J]. 中华实验和临床感染病杂志(电子版), 2021, 15(05): 295-302.
[12] 付丽华, 胡玉红, 付冬, 康晓迪, 王淑媛, 李丽. 乙型肝炎肝硬化合并妊娠对妊娠并发症及新生儿结局的影响[J]. 中华实验和临床感染病杂志(电子版), 2021, 15(04): 229-234.
[13] 陈淑钿, 梁韵, 廖媛, 王杨. 补体C3在HBV相关慢加急性肝衰竭患者预后评估中的价值[J]. 中华肝脏外科手术学电子杂志, 2023, 12(05): 562-566.
[14] 孙克彦, 毛家玺, 刘业, 刘聪, 郭闻渊, 张磊, 滕飞. HBV相关性慢加急性肝衰竭等待肝移植患者血浆置换疗效影响因素[J]. 中华肝脏外科手术学电子杂志, 2022, 11(03): 252-257.
[15] 汤永昌, 袁峰, 梁豪, 钟昭众, 熊志勇, 曹明波, 任昱朋, 李宇轩, 姚志成, 邓美海. HBx对HBV相关性肝癌增殖和迁移能力的影响及其机制[J]. 中华肝脏外科手术学电子杂志, 2022, 11(02): 198-202.
阅读次数
全文


摘要

版权所有 中华医学会 中华医学电子音像出版社有限责任公司  京ICP备14006079号-1  网络出版服务许可证：(署)网出证(京)字第075号