Taranta Andrzej, Tien Sy Bui, Zacher Behrend Johan, Rogalska-Taranta Magdalena, Manns Michael Peter, Bock Claus Thomas, Wursthorn Karsten
Hannover Medical School (MHH), Department of Gastroenterology, Hepatology and Endocrinology, Carl-Neuberg-Straße 1, 30623 Hannover, Germany.
Robert Koch Institute, Department of Infectious Diseases, Seestraße 10, 13353 Berlin, Germany; Institute of Tropical Medicine, University of Tuebingen, Wilhelmstraße 27D, 72074 Tübingen, Germany.
J Clin Virol. 2014 Aug;60(4):354-60. doi: 10.1016/j.jcv.2014.04.015. Epub 2014 May 2.
Hepatitis B virus (HBV) replicates via reverse transcription converting its partially double stranded genome into the covalently closed circular DNA (cccDNA). The long-lasting cccDNA serves as a replication intermediate in the nuclei of hepatocytes. It is an excellent, though evasive, parameter for monitoring the course of liver disease and treatment efficiency.
To develop and test a new approach for HBV DNA quantification in serum and small-size liver samples.
The p3io plasmid contains an HBV fragment and human β-actin gene (hACTB) as a standard. Respective TaqMan probes were labeled with different fluorescent dyes. A triplex real-time PCR for simultaneous quantification of total HBV DNA, cccDNA and hACTB could be established.
Three-in-one method allows simultaneous analysis of 3 targets with a lower limit of quantification of 48 copies per 20 μl PCR reaction and a wide range of linearity (R(2)>0.99, p<0.0001) for all measured sequences. The method showed a pan-genotypic specificity among genotypes A-F with serum DNA samples from HBV infected patients. Total HBV DNA and cccDNA could be quantified in 32 and 22 of 33 FFPE preserved liver specimens, respectively. Total HBV DNA concentrations quantified by the 3io method remained comparable with Cobas TaqMan HBV Test v2.0.
The three-in-one protocol allows the single step quantification of viral DNA in samples from different sources. Therefore lower sample input, faster data acquisition, a lowered error and significantly lower costs are the advantages of the method.
乙型肝炎病毒（HBV）通过逆转录进行复制，将其部分双链基因组转化为共价闭合环状DNA（cccDNA）。持久存在的cccDNA作为肝细胞细胞核中的复制中间体。它是监测肝病进程和治疗效果的一个很好但难以捉摸的参数。
开发并测试一种用于血清和小尺寸肝脏样本中HBV DNA定量的新方法。
p3io质粒包含一个HBV片段和人β-肌动蛋白基因（hACTB）作为标准。相应的TaqMan探针用不同的荧光染料标记。可以建立一种用于同时定量总HBV DNA、cccDNA和hACTB的三重实时PCR。
三合一方法允许同时分析3个靶标，每个20μl PCR反应的定量下限为48拷贝，并且对于所有测量序列具有宽线性范围（R²>0.99，p<0.0001）。该方法对来自HBV感染患者的血清DNA样本在A-F基因型之间显示出泛基因型特异性。在33个福尔马林固定石蜡包埋（FFPE）保存的肝脏标本中，分别有32个和22个标本可以定量总HBV DNA和cccDNA。通过3io方法定量的总HBV DNA浓度与Cobas TaqMan HBV Test v2.0相当。
三合一方案允许对来自不同来源的样本中的病毒DNA进行单步定量。因此，较低的样本输入量、更快的数据采集速度、更低的误差和显著更低的成本是该方法的优点。
