Detection and quantification of hepatitis B virus DNA by SYBR green real-time polymerase chain reaction

  • D. Olioso
  • M. Boaretti
  • M. Ligozzi
  • G. Lo Cascio
  • R. FontanaEmail author


A single-round real-time polymerase chain reaction (PCR) assay based on SYBR green dye technology for the detection and quantification of hepatitis B virus (HBV) DNA in serum was evaluated and compared with a qualitative nested PCR and the Cobas Amplicor HBV Monitor assay (Roche Molecular Diagnostics, Milan, Italy). The performance of the real-time PCR assay was evaluated in a routine clinical laboratory setting with a total of 212 clinical specimens. The sensitivity of the real-time PCR corresponded to 31 IU/ml (70 copies/ml), and comparison with the qualitative nested PCR showed significant concordance for 94% of samples. The linear curve over 7 log units, spanning 103–109 IU/ml (2.28 × 103 to 2.28 × 109 copies/ml), was observed in the quantitative determination. The interexperimental variability coefficient of the assay ranged from 0.22 to 0.39 and the intraexperimental variability coefficient from 0.24 to 0.41. By excluding values outside of the dynamic ranges of both tests, the HBV Monitor and the real-time PCR gave an agreement within ±1 log unit for 90% of samples, while those for the remaining 10% were found to be above 1 log unit but less than 1.5 log units. When the results inside and outside the dynamic range of the HBV Monitor were examined, 90% of the results were in agreement. In conclusion, the real-time PCR based on SYBR green technology proved suitable for routine diagnostic purposes, showing good sensitivity, high specificity, high reproducibility, and good linearity over a broad dynamic range of quantification.


Polymerase Chain Reaction Assay Nest Polymerase Chain Reaction Nest Polymerase Chain Reaction Assay Roche Molecular Diagnostics Dynamic Quantification Range 
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We wish to thank F. de Martino and V. Salotti for their help with the theoretical background of the real-time PCR technology. This study was supported by a grant from the University of Verona. D.O. is a Ph.D. fellow supported by Verona University.


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Copyright information

© Springer-Verlag 2006

Authors and Affiliations

  • D. Olioso
    • 1
  • M. Boaretti
    • 1
  • M. Ligozzi
    • 1
  • G. Lo Cascio
    • 1
    • 2
  • R. Fontana
    • 1
    • 2
    • 3
    Email author
  1. 1.Dipartimento di PatologiaSezione di Microbiologia, Università di VeronaVeronaItaly
  2. 2.Servizio di MicrobiologiaVeronaItaly
  3. 3.Department of Pathology, Microbiology SectionUniversity of VeronaVeronaItaly

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