A novel method for detection of HBVcccDNA in hepatocytes using rolling circle amplification combined with in situ PCR.
A novel method for detection of HBVcccDNA in hepatocytes using rolling circle amplification combined with in situ PCR.
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DOI：
10.1186/s12879-014-0608-y
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发表时间：
2014-12-03
影响因子：
3.7
通讯作者：
Li J
中科院分区：
文献类型：
--
作者：
Zhong Y;Hu S;Xu C;Zhao Y;Xu D;Zhao Y;Zhao J;Li Z;Zhang X;Zhang H;Li J
肝内B型肝炎病毒（HBV）共价闭合环状DNA（cccDNA）是HBV复制的原始模板。cccDNA的持续存在是HBV感染复发的原因。cccDNA的检测有助于开发针对HBV复制环节的新型抗病毒药物，减少耐药和复发，发现肝外HBV感染。原位聚合酶链反应（IS-PCR）可用于确定cccDNA在肝组织中的分布和定位，但其灵敏度和特异性较低。我们建立了一种新的检测HBV cccDNA的方法，采用滚环扩增（RCA）结合IS-PCR。26例HBV感染者的肝组织活检标本，其中慢性B型肝炎（CH B）10例，肝硬化（LC）6例，肝细胞癌（HCC）10例。设计了4对引物介导RCA，用于HBV cccDNA的第一轮特异性扩增。在RCA之前，用质粒安全的ATP依赖性DNA酶（PSAD）处理患者的肝组织切片。RCA后，通过一对标记地高辛的选择性引物通过IS-PCR进一步扩增HBV cccDNA，所述引物靶向病毒的两个直接重复区（DR 1和DR 2）之间的差距区域。19例（73.07%）HBVcccDNA表达定位于肝细胞核。与IS-PCR相比，RCA的引入提高了检测限。RCA结合IS-PCR在HCC肝组织中产生强阳性信号，尽管cccDNA拷贝数低（每个细胞2个拷贝的靶序列），同时，通过阴性对照未检测到阳性信号。RCA结合IS-PCR是一种有效可行的方法，能敏感、特异地检测低拷贝cccDNA的存在，并能反映cccDNA表达水平与肝组织病理特征的关系。本文的在线版本（doi：10.1186/s12879-014-0608-y）包含补充材料，可供授权用户使用。
Intrahepatic hepatitis B virus (HBV) covalently closed circular DNA (cccDNA) is the original template for HBV replication. The persistence of cccDNA is responsible for the recurrence of HBV infection. The detection of cccDNA can help the development of new antiviral drugs against HBV replication links, and reduce the resistance and recurrence as well as to discover extrahepatic HBV infection. In situ polymerase chain reaction (IS-PCR) can be used to determine the distribution and localization of cccDNA in liver tissues, but it is hampered by its low sensitivity and specificity. We developed a novel method to detect HBV cccDNA using rolling circle amplification (RCA) combined with IS-PCR. Biopsy liver tissues were obtained from 26 patients with HBV infection, including 10 chronic hepatitis B (CHB), 6 liver cirrhosis (LC) and 10 hepatocellular carcinoma (HCC) patients. Four pairs of primers were designed to mediating RCA for the first round amplification of HBV cccDNA specifically. The liver tissue sections from patients were treated by plasmid-safe ATP-dependent DNase (PSAD) prior to RCA. After RCA, HBV cccDNA was further amplified by a pair of selective primers labeled digoxigenin that target the gap region between the two direct repeat regions (DR1 and DR2) of the virus via IS-PCR. HBVcccDNA was expressed and located in hepatocyte nucleus in 19 patients (73.07%). Compared with the IS-PCR, the introduction of RCA increase the limit of detection. RCA combined with IS-PCR yielded strong positive signals in HCC liver tissue in spite of low copy number cccDNA (2 copies of target sequence per cell), meanwhile, no positive signal was detected via negative control. RCA combined with IS-PCR is an effective and practicable method which could detect the presence of low copy number of cccDNA sensitively and specifically, and reflect the relationship between cccDNA expression level and liver tissue pathological characteristics. The online version of this article (doi:10.1186/s12879-014-0608-y) contains supplementary material, which is available to authorized users.
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DOI：
10.2165/00066982-200106020-00010
发表时间：
2001-06-01
期刊：
MOLECULAR DIAGNOSIS
影响因子：
--
作者：
Zhang, DY;Brandwein, M;Li, HB
通讯作者：
Li, HB
影响因子：
3.1
作者：
Shieh, B;Lee, SE;Li, C
通讯作者：
Li, C
影响因子：
3.1
作者：
Singh, M;Dicaire, A;Sacks, SL
通讯作者：
Sacks, SL
影响因子：
13.5
作者：
Hosaka, Tetsuya;Suzuki, Fumitaka;Kumada, Hiromitsu
通讯作者：
Kumada, Hiromitsu
影响因子：
29.4
作者：
Werle-Lapostolle, B;Bowden, S;Zoulim, F
通讯作者：
Zoulim, F