Zhong Yanwei, Hu Shuangye, Xu Chen, Zhao Yulai, Xu Dongping, Zhao Yanqing, Zhao Jingmin, Li Zhibin, Zhang Xiuchang, Zhang Hongfei, Li Jin
Institute of Infectious Diseases, Beijing 302 Hospital, Beijing, China.
He Bei North University, Zhangjiakou, China.
BMC Infect Dis. 2014 Dec 3;14:608. doi: 10.1186/s12879-014-0608-y.
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.
肝内乙型肝炎病毒（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。检测cccDNA有助于开发针对HBV复制环节的新型抗病毒药物，降低耐药性和复发率，并发现肝外HBV感染。原位聚合酶链反应（IS-PCR）可用于确定肝组织中cccDNA的分布和定位，但因其低敏感性和特异性而受到限制。我们开发了一种结合滚环扩增（RCA）和IS-PCR检测HBV cccDNA的新方法。
从26例HBV感染患者中获取肝组织活检标本，其中包括10例慢性乙型肝炎（CHB）患者、6例肝硬化（LC）患者和10例肝细胞癌（HCC）患者。设计了四对引物介导RCA，用于第一轮特异性扩增HBV cccDNA。在进行RCA之前，用质粒安全的ATP依赖性脱氧核糖核酸酶（PSAD）处理患者的肝组织切片。RCA后，通过IS-PCR用一对标记地高辛的选择性引物进一步扩增病毒两个直接重复区域（DR1和DR2）之间间隙区域的HBV cccDNA。
19例患者（73.07%）的肝细胞核中表达并定位有HBV cccDNA。与IS-PCR相比，RCA的引入提高了检测限。尽管HCC肝组织中cccDNA拷贝数较低（每个细胞2个靶序列拷贝），但RCA与IS-PCR联合产生了强阳性信号，同时阴性对照未检测到阳性信号。
RCA与IS-PCR联合是一种有效可行的方法，可灵敏、特异地检测低拷贝数cccDNA的存在，并反映cccDNA表达水平与肝组织病理特征之间的关系。
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