Wongjitrat Chatchawal, Horthongkham Navin, Sutthent Ruengpung, Srisurapanon Surangrat
J Med Assoc Thai. 2015 Oct;98 Suppl 9:S140-5.
HBV infection causes a potential serious public health problem. The ability to detect HBV DNA concentration is an important issue that had been continuously improved. When using quantitative polymerase chain reaction (qPCR), several factors are of concern, for example, sources of material, standard curve calibration, and PCR efficiency. Digital PCR (dPCR) is an alternative PCR-based technique for absolute quantification using Poisson's statistics without requiring a standard curve.
Compare the data set of HBV DNA generated between dPCR and qPCR methods.
Fifty-four samples were quantified by Abbot's real time PCR and with 2-6 log10 HBV DNA were selected for comparison with dPCR.
Of these 54 samples, there were two outlier samples defined as negative by dPCR, whereas 52 samples were positive by both of these assays. The difference between two assays was less than 0.25 log IU/mL in 24/52 samples (46%) of paired samples; less than 0.5 log IU/mL in 46/52 samples (88%) and less than 1 log in 50/52 samples (96%). The correlation coefficient (r) was 0.788 (p-value < 0.0001). Comparison with qPCR method, data generated by dPCR tend to be an overestimation in the sample with the low level ofHBVDNA concentration and underestimated in the sample with high viral load. The variation of DNA by dPCR measurement might be due to the pre-amplification procedure and PCR template.
Measurement of HBV DNA by using dPCR, the results ofthe HBV DNA copy number tended to be deviated by over- or under-estimated when comparison to real time PCR method. In addition, a large quantity of DNA was used when compared to qPCR. However, the optimum processes of this assay have to be further investigated.
乙肝病毒（HBV）感染引发了一个潜在的严重公共卫生问题。检测HBV DNA浓度的能力是一个一直在不断改进的重要问题。使用定量聚合酶链反应（qPCR）时，有几个因素值得关注，例如材料来源、标准曲线校准和PCR效率。数字PCR（dPCR）是一种基于PCR的替代技术，用于基于泊松统计的绝对定量，无需标准曲线。
比较dPCR和qPCR方法生成的HBV DNA数据集。
通过雅培实时PCR对54个样本进行定量，并选择2 - 6 log10 HBV DNA的样本与dPCR进行比较。
在这54个样本中，有两个异常样本经dPCR定义为阴性，而这两种检测方法对另外52个样本均检测为阳性。在配对样本的52个样本中的24个（46%）中，两种检测方法之间的差异小于0.25 log IU/mL；在46/52个样本（88%）中差异小于0.5 log IU/mL，在50/52个样本（96%）中差异小于1 log。相关系数（r）为0.788（p值<0.0001）。与qPCR方法相比，dPCR生成的数据在HBV DNA浓度低的样本中往往被高估，而在病毒载量高的样本中被低估。dPCR测量的DNA变异可能归因于预扩增程序和PCR模板。
使用dPCR测量HBV DNA时，与实时PCR方法相比，HBV DNA拷贝数结果往往会出现高估或低估的偏差。此外，与qPCR相比使用了大量的DNA。然而，该检测方法的最佳流程仍需进一步研究。
