Hepatitis C is an infection caused by the hepatitis C virus (HCV). There is currently no effective HCV vaccine, so accurate diagnosis and treatment are the only methods to eliminate HCV and prevent its transmission in the community. Real-time polymerase chain reaction (real-time PCR) is the main method to quantify HCV in patient's blood; however, this method requires a standard curve to quantify the virus that may cause deviation from reality due to different replication efficiency in clinical samples. Digital PCR (dPCR) is a new developed method that can overcome the disadvantages of real-time PCR. This method allows the direct quantification of virus in patient samples without relying on standard curves, and is highly sensitive. In this study, we developed a HCV quantification assay using chip-based dPCR technology (Berto et al. 2017Kuypers and Jerome, 2017; Pasloske et al. 1998).

The 5'UTR non-encoding sequence of HCV was cloned into the BH20 plasmid to create the positive control for HCV quantification based on armored RNA technology. Using this positive control, we optimized conditions for the chip-based dPCR reaction as well as to define the limit of detection of the test. The sensitivity and specificity of the establised dPCR assay were evaluated by testing on 109 clinical samples. Finally, the assay was tested in 15 patients who were taking medication to follow up the viral titre during the treatment. All the quantification results obtained from dPCR assay were compared to those measured by COBAS AmpliPrep/COBAS TaqMan HCV Quantitative v2.0 kit (Roche).

Our developed HCV quantification assay using chip-based dPCR achieved the following standard criteria, including: (1) The assay can detect different HCV genotypes (from genotype 1 to 6) and subtypes; (2) Limit of detection (LoD) is 27.9 copies/mL, which is more sensitive than commercially available kits; (3) High accuracy with intra- and inter-coefficients of variation (CV) ≤ 1.47% and ≤ 9.59%, respectively; (4) Sensitivity is 100%, specificity is 98%; (5) Quantitative results of RNA HCV in 109 clinical samples were similar to those measured with the standard COBAS AmpliPrep/COBAS TaqMan HCV Quantitative v2.0 kit (Roche). Moreover, we noted that dPCR could quantify well samples with low viral titre (< 100 copies/mL).

The developed chip-based dPCR assay can be applied in the quantification of HCV with high accuracy and sensitivity.

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