Real-time PCR assay for detection and quantification of hepatitis B virus genotypes A to G

Abstract

The detection and quantification of hepatitis B virus (HBV) DNA play an important role in diagnosing and monitoring HBV infection as well as assessing therapeutic response. The great variability among HBV genotypes and the enormous range of clinical HBV DNA levels present challenges for PCR-based amplification techniques. In this study, we describe the development, evaluation, and validation of a novel real-time PCR assay designed to provide accurate quantification of DNA from all eight HBV genotypes in patient plasma specimens. A computer algorithm was used to design degenerate real-time PCR primers and probes based upon a large number (n = 340) of full-length genomic sequences including HBV genotypes A to H from Europe, Africa, Asia, and North and South America. Genotype performance was tested and confirmed using 59 genotype A to G specimens from two commercially available worldwide genotype panels. This assay has a dynamic range of at least 8 log(10) without the need for specimen dilution, good clinical intra- and interassay precision, and excellent correlation with the Bayer Diagnostics VERSANT HBV DNA 3.0 (branched DNA) assay (r = 0.93). Probit analysis determined the 95% detection level was 56 IU/ml, corresponding to 11 copies per PCR well. The high sensitivity, wide linear range, good reproducibility, and genotype inclusivity, combined with a small sample volume requirement and low cost, make this novel quantitative HBV real-time PCR assay particularly well suited for application to large clinical and epidemiological studies.

FIG.1.

(A) HBV real-time PCR assay amplification plot obtained from triplicates of 8-log₁₀ serial dilutions of plasmid pAM6 ranging from a nominal concentration of 5 × 10¹ to 5 × 10⁸ IU/ml. This figure was generated by the ABI Prism 7700 Sequence Detection Software (SDS) version 1.9.1. (B) HBV real-time PCR plasmid pAM6 standard curve showing linearity over an 8-log₁₀ dynamic range. This standard curve was generated from the amplification plot displayed in Fig. 1A. The least-squares regression was calculated from plots of measured C_T (y axis) versus input plasmid DNA over a range of 5 × 10¹ to 5 × 10⁸ IU/ml (x axis) tested in triplicate PCR wells per dilution. The correlation coefficient was 0.997, and the slope of the line was −3.36.

FIG.1.

(A) HBV real-time PCR assay amplification plot obtained from triplicates of 8-log₁₀ serial dilutions of plasmid pAM6 ranging from a nominal concentration of 5 × 10¹ to 5 × 10⁸ IU/ml. This figure was generated by the ABI Prism 7700 Sequence Detection Software (SDS) version 1.9.1. (B) HBV real-time PCR plasmid pAM6 standard curve showing linearity over an 8-log₁₀ dynamic range. This standard curve was generated from the amplification plot displayed in Fig. 1A. The least-squares regression was calculated from plots of measured C_T (y axis) versus input plasmid DNA over a range of 5 × 10¹ to 5 × 10⁸ IU/ml (x axis) tested in triplicate PCR wells per dilution. The correlation coefficient was 0.997, and the slope of the line was −3.36.

FIG. 2.

Clinical linear dynamic range of the HBV real-time PCR assay. These data were obtained using a 7-log₁₀ dilution series of a high-titer HBV genotype A plasma specimen in eight independent experiments. Each dilution was tested in triplicate, resulting in a total of 24 replicate measurements. The assay is linear over a range of 2.88 × 10⁰ to at least 2.69 × 10⁷ IU/ml.

FIG. 3.

Graph showing correlation of the HBV real-time PCR assay results with the Bayer Diagnostics VERSANT HBV DNA 3.0 (bDNA) assay results for 29 HBsAg-positive clinical specimens (numerical values are listed in boldface in Table 3). The HBV genotype of each specimen is adjacent to the data point on the graph. There was excellent correlation (r = 0.93) between the two assays for all HBV genotypes (A to G).

FIG. 4.

Bar graph showing QC tracking of HBV real-time PCR positive control results over time. The BBI ACCURUN positive control was extracted and tested (in triplicate) concurrently with samples in 78 independent experiments over the course of 1 year. Each vertical bar represents the average positive control result for a single experiment. The overall average is shown as a horizontal black line. The positive control QC upper and lower limits, shown as horizontal red lines, were calculated as 2 SD above and below the average, respectively. Only 3 of the 78 positive control results (3.8%) exceeded these limits.