Comparison of droplet digital PCR and real time PCR method for HBV DNA quantification

Abstract

Background: 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. Objective: Compare the data set of HBV DNA generated between dPCR and qPCR methods. Material and 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. Results: 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 of HBV DNA 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. Conclusion: Measurement of HBV DNA by using dPCR, the results of the 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. © 2015, Medical Association of Thailand. All rights reserved.