Exonuclease I and III improve the detection efficacy of hepatitis B virus covalently closed circular DNA
Exonuclease I and III improve the detection efficacy of hepatitis B virus covalently closed circular DNA
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核酸外切酶 I 和 III 提高了乙型肝炎病毒共价闭合环状 DNA 的检测效率。
DOI：
10.1016/j.hbpd.2018.11.003
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发表时间：
2019-10-01
影响因子：
3.3
通讯作者：
Zhang, Ji-Ming
中科院分区：
文献类型：
--
作者：
Jiang, Pei-Xue;Mao, Ri-Cheng;Zhang, Ji-Ming
背景：B型肝炎病毒共价闭合环状DNA（HBV cccDNA）是检测B型肝炎病毒感染的重要生物标志物。然而，目前的方法特异性和敏感性不高。方法：采用核酸外切酶I（ExoI）、核酸外切酶III（ExoIII）和特异性引物探针，建立实时荧光定量PCR（real-time PCR）方法。以外周血单个核细胞分离的病毒颗粒为阴性对照，HBV1.3重组质粒3.2kb环状DNA片段为阳性对照。结果：HBV cccDNA特异性引物检测cccDNA的线性范围为10（1）~ 10（7）拷贝/次。HBV cccDNA仅在细胞系、动物模型和肝组织中检测到。在血清样本中无法检出。结论：实时荧光定量PCR是检测低拷贝HBV cccDNA的一种有效、可行的方法。该检测方法快速、灵敏度高、特异性强，可用于临床。(C)2018年中国浙江大学医学院附属第一医院。Elsevier B. V.出版，保留所有权利。
Background: Hepatitis B virus covalently closed circular DNA (HBV cccDNA) is an important biomarker of hepatitis B virus infection. However, the current methods are not specific and sensitive. The present study aimed to develop a specific and sensitive assay method for the quantification of HBV cccDNA.Methods: Exonuclease I (Exo I) & Exonuclease III (Exo III) and specific primer probes are used in real-time PCR. The virus particles isolated from peripheral blood mononuclear cells were used as negative control and HBV1.3 recombinant plasmid 3.2 kb circular DNA fragment was used as positive control. The methods of cccDNA detection were evaluated in cell lines, plasmid, animal model, patient serum and liver biopsies.Results: A linear range of 10(1)-10(7) copies/assay using specific primers for HBV cccDNA was established. HBV cccDNA were only detected in cell lines, animal model and liver tissue. It cannot be detected in serum samples. Intrahepatic HBV cccDNA level had good correlation with intrahepatic total HBV DNA level (r = 0.765, P < 0.001).Conclusions: The real-time quantitative PCR is an effective and feasible method for sensitive and specific detection of low copy number of cccDNA. The novel detection method is fast, provides high sensitivity and specificity and can be used in clinical practice. (C) 2018 First Affiliated Hospital, Zhejiang University School of Medicine in China. Published by Elsevier B.V. All rights reserved.