摘要
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HindⅢ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。
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).MethodsAfter 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 withHindⅢendonuclease 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.ResultsAlpha-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.ConclusionsA 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.
作者
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
Junwen Wang;Yuan Tian;Zihao Fan;Ling Xu;Yao Gao;Yaling Cao;Zhenzhen Pan;Xiangying Zhang;Yan Song;Feng Ren(Department of Clinical Laboratory,Beijing Chuiyangliu Hospital,Beijing 100022,China;Beijing Institute of Hepatology,Beijing Youan Hospital,Capital Medical University,Beijing 100069,China)
出处
《中华实验和临床感染病杂志（电子版）》
CAS
2022年第5期320-327,共8页
Chinese Journal of Experimental and Clinical Infectious Diseases(Electronic Edition)
基金
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)
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
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
作者简介
通信作者:任锋,Email:renfeng7512@hotmail.com
