1887

Journal of Medical Microbiology logo

Volume 52, Issue 5

Rapid quantification of hepatitis B virus DNA by real-time PCR using fluorescent hybridization probes Free

Abstract

A highly sensitive and rapid assay has been developed to quantify hepatitis B virus (HBV) DNA, based on the fluorescence resonance energy transfer principle and real-time PCR, using the LightCycler and a pair of specific fluorescent hybridization probes. This LightCycler real-time PCR assay (LC-PCR) detected HBV DNA in a linear range from 10 to 10 copies per reaction (250–2.5 × 10 copies ml), with a rapid PCR cycling time of 35 min. The assay was validated with two EUROHEP HBV DNA standards ( and subtypes) and exhibited low intra-assay (< 6 %) and inter-assay (< 16 %) variation for both subtypes over the complete range of 7 orders of magnitude. The assay was evaluated clinically using serum samples from 120 HBsAg individuals and 45 healthy controls who were negative for both HBsAg and anti-HBc. Levels of HBV DNA were measured in these samples using both the LC-PCR and Digene Hybrid Capture II HBV DNA (HCII) assays. The prevalence rates for HBV DNA in the HBsAg serum samples were respectively 95 % (114/120) and 56 % (67/120) by LC-PCR and HCII ( < 0.01). All 67 HCII-positive samples tested positive with LC-PCR, while the 47 discordant samples showed low levels of HBV DNA (down to 265 copies ml), detectable only by the more sensitive LC-PCR assay. Levels of HBV DNA as measured by the two assays showed good correlation ( = 0.902; < 0.001). The level of HBV DNA was significantly higher in HBeAg than anti-HBe samples (median 1.5 × 10 vs 4.6 × 10 copies ml; < 0.01). It is concluded that this LC-PCR assay is clinically useful for the rapid, sensitive and accurate measurement of HBV DNA.

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© Microbiology Society
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2003-05-01
2023-04-01
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References

  1. Abe A, Inoue K, Tanaka T, Kato J, Kajiyama N, Kawaguchi R, Tanaka S, Yoshiba M, Kohara M. 1999; Quantitation of hepatitis B virus genomic DNA by real-time detection PCR. J Clin Microbiol 37:2899–2903
     [Google Scholar]
  2. Brechtbuehl K, Whalley S. A, Dusheiko G. M, Saunders N. A. 2001; A rapid real-time quantitative polymerase chain reaction for hepatitis B virus. J Virol Methods 93:105–113 [CrossRef]
     [Google Scholar]
  3. Cane P. A, Cook P, Ratcliffe D, Mutimer D, Pillay D. 1999; Use of real-time PCR and fluorimetry to detect lamivudine resistance- associated mutations in hepatitis B virus. Antimicrob Agents Chemother 43:1600–1608
     [Google Scholar]
  4. Chan T. M, Wu P. C, Li F. K, Lai C. L, Cheng I. K, Lai K. N. 1998; Treatment of fibrosing cholestatic hepatitis with lamivudine. Gastroenterology 115:177–181 [CrossRef]
     [Google Scholar]
  5. Chan T. M, Fang G. X, Tang C. S. O, Cheng I. K. P, Lai K. N, Ho S. K. N. 2002; Preemptive lamivudine therapy based on HBV DNA level in HBsAg-positive kidney allograft recipients. Hepatology 36:1246–1252 [CrossRef]
     [Google Scholar]
  6. Chung H. T, Lok A. S, Lai C. L. 1993; Re-evaluation of alpha-interferon treatment of chronic hepatitis B using polymerase chain reaction. J Hepatol 17:208–214 [CrossRef]
     [Google Scholar]
  7. Espy M. J, Uhl J. R, Mitchell P. S, Thorvilson J. N, Svien K. A, Wold A. D, Smith T. F. 2000; Diagnosis of herpes simplex virus infections in the clinical laboratory by LightCycler PCR. J Clin Microbiol 38:795–799
     [Google Scholar]
  8. Ghosh S. S, Eis P. S, Blumeyer K, Fearon K, Millar D. P. 1994; Real time kinetics of restriction endonuclease cleavage monitored by fluorescence resonance energy transfer. Nucleic Acids Res 22:3155–3159 [CrossRef]
     [Google Scholar]
  9. Heermann K. H, Gerlich W. H, Chudy M, Schaefer S, Thomssen R. 1999; Quantitative detection of hepatitis B virus DNA in two international reference plasma preparations.Eurohep Pathobiology Group. J Clin Microbiol 37:68–73
     [Google Scholar]
  10. Hendricks D. A, Stowe B. J, Hoo B. S, Kolberg J, Irvine B. D, Neuwald P. D, Urdea M. S, Perrillo R. P. 1995; Quantitation of HBV DNA in human serum using a branched DNA (bDNA) signal amplification assay. Am J Clin Pathol 104:537–546
     [Google Scholar]
  11. Higuchi R, Dollinger G, Walsh P. S, Griffith R. 1992; Simultaneous amplification and detection of specific DNA sequences. Biotechnology 10:413–417 [CrossRef]
     [Google Scholar]
  12. Ho S. K, Chan T. M. 2000; An overview of assays for serum HBV DNA. Clin Lab 46:609–614
     [Google Scholar]
  13. Ho S. K, Chan T. M, Cheng I. K, Lai K. N. 1999; Comparison of the second-generation digene hybrid capture assay with the branched-DNA assay for measurement of hepatitis B virus DNA in serum. J Clin Microbiol 37:2461–2465
     [Google Scholar]
  14. Holland P. M, Abramson R. D, Watson R, Gelfand D. H. 1991; Detection of specific polymerase chain reaction product by utilizing the 5′–3′ exonuclease activity of Thermus aquaticus DNA polymerase. Proc Natl Acad Sci U S A 88:7276–7280 [CrossRef]
     [Google Scholar]
  15. Jardi R, Rodriguez F, Buti M, Costa X, Cotrina M, Valdes A, Galimany R, Esteban R, Guardia J. 2001; Quantitative detection of hepatitis B virus DNA in serum by a new rapid real-time fluorescence PCR assay. J Viral Hepat 8:465–471 [CrossRef]
     [Google Scholar]
  16. Kaneko S, Miller R. H, Di Bisceglie A. M, Feinstone S. M, Hoofnagle J. H, Purcell R. H. 1990; Detection of hepatitis B virus DNA in serum by polymerase chain reaction.Application for clinical diagnosis. Gastroenterology 99:799–804
     [Google Scholar]
  17. Kessler H. H, Pierer K, Dragon E, Lackner H, Santner B, Stunzner D, Stelzl E, Waitzl B, Marth E. 1998; Evaluation of a new assay for HBV DNA quantitation in patients with chronic hepatitis B. Clin Diagn Virol 9:37–43 [CrossRef]
     [Google Scholar]
  18. Lai C. L, Chien R. N, Leung N. W. 9 other authors 1998; A one-year trial of lamivudine for chronic hepatitis B.Asia Hepatitis Lamivudine Study Group. N Engl J Med 339:61–68 [CrossRef]
     [Google Scholar]
  19. Lai V. C, Guan R, Wood M. L, Lo S. K, Yuen M. F, Lai C. L. 1999; Nucleic acid-based cross-linking assay for detection and quantification of hepatitis B virus DNA. J Clin Microbiol 37:161–164
     [Google Scholar]
  20. Lee W. M. 1997; Hepatitis B virus infection. N Engl J Med 337:1733–1745 [CrossRef]
     [Google Scholar]
  21. Loeb K. R, Jerome K. R, Goddard J, Huang M, Cent A, Corey L. 2000; High-throughput quantitative analysis of hepatitis B virus DNA in serum using the TaqMan fluorogenic detection system. Hepatology 32:626–629 [CrossRef]
     [Google Scholar]
  22. Niesters H. G, Krajden M, Cork L, de Medina M, Hill M, Fries E, Osterhaus A. D. 2000; A multicenter study evaluation of the digene hybrid capture II signal amplification technique for detection of hepatitis B virus DNA in serum samples and testing of EUROHEP standards. J Clin Microbiol 38:2150–2155
     [Google Scholar]
  23. Noborg U, Gusdal A, Pisa E. K, Hedrum A, Lindh M. 1999; Automated quantitative analysis of hepatitis B virus DNA by using the Cobas Amplicor HBV monitor test. J Clin Microbiol 37:2793–2797
     [Google Scholar]
  24. Omata M. 1998; Treatment of chronic hepatitis B infection. N Engl J Med 339:114–115 [CrossRef]
     [Google Scholar]
  25. Paraskevis D, Haida C, Tassopoulos N, Raptopoulou M, Tsantoulas D, Papachristou H, Sypsa V, Hatzakis A. 2002; Development and assessment of a novel real-time PCR assay for quantitation of HBV DNA. J Virol Methods 103:201–212 [CrossRef]
     [Google Scholar]
  26. Pas S. D, Fries E, De Man R. A, Osterhaus A. D, Niesters H. G. 2000; Development of a quantitative real-time detection assay for hepatitis B virus DNA and comparison with two commercial assays. J Clin Microbiol 38:2897–2901
     [Google Scholar]
  27. Puchhammer-Stockl E, Mandl C. W, Kletzmayr J, Holzmann H, Hofmann A, Aberle S. W, Heinz F. X, Watschinger B, Hofmann H. 2000; Monitoring the virus load can predict the emergence of drug-resistant hepatitis B virus strains in renal transplantation patients during lamivudine therapy. J Infect Dis 181:2063–2066 [CrossRef]
     [Google Scholar]
  28. Quint W. G, Heijtink R. A, Schirm J, Gerlich W. H, Niesters H. G. 1995; Reliability of methods for hepatitis B virus DNA detection. J Clin Microbiol 33:225–228
     [Google Scholar]
  29. Weinberger K. M, Wiedenmann E, Bohm S, Jilg W. 2000; Sensitive and accurate quantitation of hepatitis B virus DNA using a kinetic fluorescence detection system (TaqMan PCR). J Virol Methods 85:75–82 [CrossRef]
     [Google Scholar]
  30. Whalley S. A, Brown D, Teo C. G, Dusheiko G. M, Saunders N. A. 2001; Monitoring the emergence of hepatitis B virus polymerase gene variants during lamivudine therapy using the LightCycler. J Clin Microbiol 39:1456–1459 [CrossRef]
     [Google Scholar]
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Rapid quantification of hepatitis B virus DNA by real-time PCR using fluorescent hybridization probes
J. Med. Microbiol. 52, 397 (2003); https://doi.org/10.1099/jmm.0.05071-0
/content/journal/jmm/10.1099/jmm.0.05071-0
/content/journal/jmm/10.1099/jmm.0.05071-0
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