A Highly Sensitive and Robust Method for Hepatitis B Virus Covalently Closed Circular DNA Detection in Single Cells and Serum

作者：Huang, Jing-Tao; Yang, Ying; Hu, Yi-Min; Liu, Xing-Hui; Liao, Mei-Yan; Morgan, Roy; Yuan, Er-Feng; Li, Xia; Liu, Song-Mei^*

来源：Journal of Molecular Diagnostics, 2018, 20(3): 334-343.

DOI：10.1016/j.jmoldx.2018.01.010

• DROPLET DIGITAL PCR
• HEPATOCELLULAR-CARCINOMA
• CYSTATIN-C
• CANCER PATIENTS
• HEPATOCYTES
• THERAPY
• CCCDNA
• INFECTION
• QUANTIFICATION
• QUANTITATION

Despite implications of persistence of hepatitis B virus (HBV) covalently closed circular DNA (cccDNA) in the development of hepatocellular carcinoma (HCC), little is known about serum cccDNA in HBV-infected diseases. We developed a cccDNA-selective droplet digital PCR (ddPCR) to assess cccDNA content and dynamics across different stages of HCC development. One hundred forty-seven serum samples and 35 formalin-fixed, paraffin-embedded tumor tissues were derived from patients with HCC or HBV hepatitis/cirrhosis. After specific amplification and selective digestion, probe-based ddPCR was used to quantify cccDNA copy numbers in single cells and clinical samples. The cccDNA in single HepG2.2.15 cells ranged from 0 to 10.8 copies/cell. Compared with non-HCC patients, HCC patients showed a higher cccDNA-positive rate (89.9% versus 53.2%; P = 4.22 x 10(-6)) and increased serum cccDNA contents (P = 0.002 and P = 0.041 for hepatitis and cirrhosis patients, respectively). Serum cccDNA ranged from 84 to 1.07 x 10(5) copies/mL. Quantification of serum cccDNA and HBV-DNA was an effective way to discriminate HCC patients from non-HCC patients, with areas under the curve of receiver operating characteristic of 0.847 (95 % CI, 0.759-0.935; sensitivity, 74.5 %; specificity, 93.7%). cccDNA-selective ddPCR is sensitive to detect cccDNA in single cells and different clinical samples. Combined analysis of serum cccDNA and HBV-DNA may be a promising strategy for HBV-induced HCC surveillance and antiviral therapy evaluation.

• 出版日期2018-5
• 单位湖南省人民医院; 中国人民解放军第二军医大学; 武汉市医疗救治中心; 上海市浦东新区公利医院; 武汉大学