Rapid quantification assay of hepatitis B virus DNA in human serum and plasma by Fully Automated Genetic Analyzer μTASWako g1

Abstract

Real-time monitoring of serum hepatitis B virus (HBV) levels is essential for the management of patients with chronic HBV infection in clinical practice, including monitoring the resistance of anti-HBV nucleotide analog or the detection of HBV reactivation. In this context, serum HBV deoxyribonucleic acid (DNA) quantification should be rapidly measured. A rapid HBV DNA quantification assay was established on the Fully Automated Genetic Analyzer, μTASWako g1. The assay performs automated sample preparation and DNA extraction, followed by the amplification and detection of quantitative polymerase chain reaction (PCR) combined with capillary electrophoresis (qPCR-CE) on integrated microfluidic chip. This study aimed to evaluate the analytical and clinical performance of HBV DNA assay on the μTASWako g1 platform in human serum and EDTA-plasma. The HBV DNA assay has a linear quantitative range from 20 to 108 IU/mL of HBV DNA with standard deviation (SD) of ≤0.14 log10 IU/mL. The limits of detection of the assay were 4.18 for the serum and 4.35 for EDTA-plasma. The HBV assay demonstrated the equivalent performance in both human serum and EDTA-plasma matrices. The HBV genotypes A to H were detected with an accuracy of ±0.34 log10 IU/mL. In quantification range, the HBV DNA assay was correlated with Roche cobas AmpliPrep/cobas TaqMan Ver2.0 (CAP/CTM v2) (r = 0.964). The mean difference (μTASWako g1-CAP/CTM v2) of the reported HBV DNA was -0.01 log10 IU/mL. Overall, the sensitivity, accuracy, and precision of the μTASWako g1 HBV assay were comparable to the existing commercial HBV DNA assay, and the assay can be completed within 110 min. This evaluation suggests that the HBV DNA assay on the μTASWako g1 is potentially applied for alternative method of the HBV viral load test, in particular with the advantage of the HBV DNA result availability within 2 h, improving the HBV infection management.

Fig 1. Real-time detection of HBV target using the μTASWako g1 analyzer.

(a) Overlay of electropherograms in the PCR-CE for 10⁴ IU/mL of the 4^th WHO International Standard for HBV. Merged images of 33 electropherograms from 8^th to 40^th PCR cycle. (b) HBV amplification growth curves. RFU stands for relative fluorescence unit.

Fig 2. HBV Cq and IC Cq values plot.

3.5 ×10⁸, 10⁸, 10⁷, 10⁶, 10⁵, 10⁴, 10³, 10², and 10 IU/mL of the HBV-positive control in the serum were tested using the μTASWako g1 Analyzer. One replicate for each concentration. PCR efficiency of HBV target amplification was 98.8%.

Fig 3. Traceability to the WHO international standard and matrix equivalency between serum and plasma on μTASWako g1 analyzer.

Dilutions of the positive control (PC) and WHO international standard (WHO std) were tested in three replicates for each concentration. The dots represent the mean log₁₀ transformed titer. The dashed line represents the equality line.

Fig 4. Correlation of measurement and limit of agreement between HBV assays on the μTASWako g1 analyzer and CAP/CTM v2.

A total of 157 serum samples from HBV-infected patients were tested with CAP/CTM v2 and μTASWako g1 Analyzer. (a) Passing–Bablok regression fit (Solid line). Correlation coefficiency (r) was 0.964 (95% CI, 0.959–0.968), P < 0.0001. The dashed line represents the equality line. (b) Bland–Altman plot shows the mean difference between CAP/CTM v2 and μTASWako g1 Analyzer was −0.01 (limit of agreement, −0.82 to −0.85 log₁₀ IU/mL). The solid and dashed lines represent the mean difference and mean ±1.96 SD, respectively.