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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
Article

Abstract

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.

Keywords

Polymerase Chain Reaction Assay Nest Polymerase Chain Reaction Nest Polymerase Chain Reaction Assay Roche Molecular Diagnostics Dynamic Quantification Range 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Notes

Acknowledgements

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.

References

  1. 1.
    Lai CL, Ratziu V, Yuen M, Poynard T (2003) Viral hepatitis B. Lancet 362:2089–2094PubMedCrossRefGoogle Scholar
  2. 2.
    Pawlotsky JM (2002) Molecular diagnosis of viral hepatitis. Gastroenterology 122:1554–1568PubMedCrossRefGoogle Scholar
  3. 3.
    Brechot C (1993) Polymerase chain reaction for the diagnosis of hepatitis B and C viral hepatitis. J Hepatol 17 (Suppl 3):S35–S41PubMedCrossRefGoogle Scholar
  4. 4.
    Kaneko S, Feinstone SM, Miller RH (1989) Rapid and sensitive method for the detection of serum hepatitis B virus DNA using the polymerase chain reaction technique. J Clin Microbiol 27:1930–1933PubMedGoogle Scholar
  5. 5.
    Valentine-Thon E, Van Loon AM, Schirm J, Reid J, Klapper PE, Cleator GM (2001) European proficiency testing program for molecular detection and quantitation of hepatitis B virus DNA. J Clin Microbiol 39:4407–4412PubMedCrossRefGoogle Scholar
  6. 6.
    Abe A, Inoue K, Tanaka T, Kato J, Kajiyama N, Kawaguchi R, Tanaka S, Yoshiba M, Kohara M (1999) Quantification of hepatitis B virus genomic DNA by real-time detection PCR. J Clin Microbiol 37:2899–2903PubMedGoogle Scholar
  7. 7.
    Komurian-Pradel F, Paranhos-Baccalà G, Sodoyer M, Chevallier P, Mandrand B, Lotteau V, Andrè P (2001) Quantitation of HCV RNA using real-time PCR and fluorimetry. J Virol Methods 95:111–119PubMedCrossRefGoogle Scholar
  8. 8.
    Nozaki A, Kato N (2002) Quantitative method of intracellular hepatitis C virus RNA using LightCycler PCR. Acta Med Okayama 56:107–110PubMedGoogle Scholar
  9. 9.
    Paraskevis D, Haida C, Tasopoulos N, Raptopoulou M, Tsantoulas D, Papachristou H, Sypsa V, Hatzakis A (2002) Development and assessment of a novel real-time PCR assay for quantification of HBV DNA. J Virol Methods 103:201–212PubMedCrossRefGoogle Scholar
  10. 10.
    Matsumoto C, Nishioka K, Oguchi T, Mitsunaga S, Nojiri N, Tadokoro K, Juji T (1997) Detection and quantitation of HBV DNA by semi-nested PCR in donated blood: comparison with HBV serological markers. J Virol Methods 66:61–69PubMedCrossRefGoogle Scholar
  11. 11.
    Chu C, Hussain M, Lok ASF (2002) Quantitative serum HBV DNA levels during different stages of chronic hepatitis B infection. Hepatology 36:1408–1415PubMedGoogle Scholar
  12. 12.
    Stelzl E, Muller Z, Marth E, Keßler HH (2004) Rapid quantification of hepatitis B virus DNA by automated sample preparation and real-time PCR. J Clin Microbiol 42:2445–2449PubMedCrossRefGoogle Scholar
  13. 13.
    Weiss J, Wu H, Farrenkopf B, Schultz T, Song G, Shah S, Siegel J (2004) Real time TaqMan PCR detection and quantitation of HBV genotypes A–G with the use of an internal quantitation standard. J Clin Virology 30:86–93CrossRefGoogle Scholar
  14. 14.
    Kidd-Ljunggren K, Miyakawa Y, Kidd AH (2002) Genetic variability in hepatitis B viruses. J Gen Virol 83:1267–1280PubMedGoogle Scholar
  15. 15.
    Kidd-Ljunggren K, Myhre E, Bläckberg J (2004) Clinical and serological variation between patients infected with different hepatitis B virus genotypes. J Clin Microbiol 42:5837–5841PubMedCrossRefGoogle Scholar

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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