外切核酸酶I和III提高了乙型肝炎病毒共价闭合环状DNA的检测

StephenW

Hepatobiliary Pancreat Dis Int. 2018 Nov 22. pii: S1499-3872(18)30261-3. doi: 10.1016/j.hbpd.2018.11.003. [Epub ahead of print]
Exonuclease I and III improve the detection efficacy of hepatitis B virus covalently closed circular DNA.
Jiang PX1, Mao RC1, Dong MH1, Yu XP1, Xun Q1, Wang JY1, Jing L1, Qiang D2, Zhang JM3.
Author information

1
    Department of Infectious Diseases, Huashan Hospital, Fudan University, Shanghai, China.
2
    Department of Pathobiology and Key Laboratory of Medical Molecular Virology, School of Basic Medical Sciences, Fudan University, Shanghai, China.
3
    Department of Infectious Diseases, Huashan Hospital, Fudan University, Shanghai, China. Electronic address: [email protected].

Abstract
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 101-107 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.

StephenW

肝胆胰疾病2018年11月22日.pii：S1499-3872（18）30261-3。 doi：10.1016 / j.hbpd.2018.11.003。 [提前打印]
外切核酸酶I和III提高了乙型肝炎病毒共价闭合环状DNA的检测效率。
Jiang PX1，Mao RC1，Dong MH1，Yu XP1，Xun Q1，Wang JY1，Jing L1，Qiang D2，Zhang JM3。
作者信息

1
    复旦大学附属华山医院感染科，上海
2
    复旦大学基础医学院病理学系，医学分子病毒学重点实验室，上海
3
    复旦大学附属华山医院感染科，上海电子地址：[email protected]。

抽象
背景：

乙型肝炎病毒共价闭合环状DNA（HBV cccDNA）是乙型肝炎病毒感染的重要生物标志物。但是，目前的方法并不具体和敏感。本研究旨在开发一种特异性和灵敏的HBV cccDNA定量分析方法。
方法：

外切核酸酶I（Exo I）和外切核酸酶III（Exo III）和特异性引物探针用于实时PCR。从外周血单核细胞分离的病毒颗粒用作阴性对照，HBV1.3重组质粒3.2kb环状DNA片段用作阳性对照。在细胞系，质粒，动物模型，患者血清和肝脏活组织检查中评估cccDNA检测方法。
结果：

建立了使用HBV cccDNA特异性引物的101-107拷贝/测定的线性范围。仅在细胞系，动物模型和肝组织中检测到HBV cccDNA。它不能在血清样本中检测到。肝内HBV cccDNA水平与肝内总HBV DNA水平有良好的相关性（r = 0.765，P <0.001）。
结论：

实时定量PCR是一种有效可行的方法，用于敏感和特异性检测低拷贝数的cccDNA。该新型检测方法快速，灵敏度高，特异性强，可用于临床。