Digoxigenin-labeled probes for the detection of hepatitis B virus DNA in serum (original) (raw)

Abstract

A nonradioactive hybridization assay for the detection of hepatitis B virus (HBV) DNA in serum with a digoxigenin-labeled probe is described. The probe was sensitive, being able to detect 0.25 pg of homologous HBV DNA, equivalent to 7 x 10(4) genome copies. After extraction of DNA from clinical samples, the probe detected HBV DNA in 11 of 12 hepatitis B e antigen-positive sera and did not react with 6 hepatitis B surface antigen-negative sera. This result was comparable to that obtained with a radiolabeled probe. When serum samples were treated by the alkaline denaturation method, some false-positive reactions were apparent with the digoxigenin-labeled probe, although their frequency could be reduced to around 8% by modifying the sample treatment with a centrifugation step. Overall, the sensitivity and specificity of the digoxigenin-labeled probe indicate that it is a viable alternative to the radiolabeled probe for the detection of HBV DNA in serum. The lack of radioactive reagents in the digoxigenin labeling and detection system and its long shelf-life make this system suitable for routine use in laboratories.

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Selected References

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  1. Berninger M., Hammer M., Hoyer B., Gerin J. L. An assay for the detection of the DNA genome of hepatitis B virus in serum. J Med Virol. 1982;9(1):57–68. doi: 10.1002/jmv.1890090109. [DOI] [PubMed] [Google Scholar]
  2. Bonino F., Hoyer B., Nelson J., Engle R., Verme G., Gerin J. Hepatitis B virus DNA in the sera of HBsAg carriers: a marker of active hepatitis B virus replication in the liver. Hepatology. 1981 Sep-Oct;1(5):386–391. doi: 10.1002/hep.1840010503. [DOI] [PubMed] [Google Scholar]
  3. Brechot C., Hadchouel M., Scotto J., Degos F., Charnay P., Trepo C., Tiollais P. Detection of hepatitis B virus DNA in liver and serum: a direct appraisal of the chronic carrier state. Lancet. 1981 Oct 10;2(8250):765–768. doi: 10.1016/s0140-6736(81)90182-3. [DOI] [PubMed] [Google Scholar]
  4. Casacuberta J. M., Jardi R., Buti M., Puigdoménech P., San Segundo B. Comparison of different non-isotopic methods for hepatitis B virus detection in human serum. Nucleic Acids Res. 1988 Dec 23;16(24):11834–11834. doi: 10.1093/nar/16.24.11834. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Gerber M. A., Thung S. N. Molecular and cellular pathology of hepatitis B. Lab Invest. 1985 Jun;52(6):572–590. [PubMed] [Google Scholar]
  6. Hoofnagle J. H., Schafer D. F. Serologic markers of hepatitis B virus infection. Semin Liver Dis. 1986 Feb;6(1):1–10. doi: 10.1055/s-2008-1040787. [DOI] [PubMed] [Google Scholar]
  7. Kaneko S., Miller R. H., Feinstone S. M., Unoura M., Kobayashi K., Hattori N., Purcell R. H. Detection of serum hepatitis B virus DNA in patients with chronic hepatitis using the polymerase chain reaction assay. Proc Natl Acad Sci U S A. 1989 Jan;86(1):312–316. doi: 10.1073/pnas.86.1.312. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Kimpton C. P., Corbitt G., Morris D. J. Detection of cytomegalovirus DNA using probes labelled with digoxigenin. J Virol Methods. 1989 Jun;24(3):335–346. doi: 10.1016/0166-0934(89)90046-3. [DOI] [PubMed] [Google Scholar]
  9. Larzul D., Guigue F., Sninsky J. J., Mack D. H., Bréchot C., Guesdon J. L. Detection of hepatitis B virus sequences in serum by using in vitro enzymatic amplification. J Virol Methods. 1988 Jul;20(3):227–237. doi: 10.1016/0166-0934(88)90126-7. [DOI] [PubMed] [Google Scholar]
  10. Lieberman H. M., LaBrecque D. R., Kew M. C., Hadziyannis S. J., Shafritz D. A. Detection of hepatitis B virus DNA directly in human serum by a simplified molecular hybridization test: comparison to HBeAg/anti-HBe status in HBsAg carriers. Hepatology. 1983 May-Jun;3(3):285–291. doi: 10.1002/hep.1840030302. [DOI] [PubMed] [Google Scholar]
  11. Saldanha J. A., Karayiannis P., Thomas H. C., Monjardino J. P. Use of biotinylated probes in serum hepatitis B virus DNA detection. J Virol Methods. 1987 Jul;16(4):339–342. doi: 10.1016/0166-0934(87)90019-x. [DOI] [PubMed] [Google Scholar]
  12. Scotto J., Hadchouel M., Hery C., Yvart J., Tiollais P., Brechot C. Detection of hepatitis B virus DNA in serum by a simple spot hybridization technique: comparison with results for other viral markers. Hepatology. 1983 May-Jun;3(3):279–284. doi: 10.1002/hep.1840030301. [DOI] [PubMed] [Google Scholar]
  13. Tenover F. C. Diagnostic deoxyribonucleic acid probes for infectious diseases. Clin Microbiol Rev. 1988 Jan;1(1):82–101. doi: 10.1128/cmr.1.1.82. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Theilmann L., Fischer M., Galle P. R., Nassal M. Detection of HBV DNA in HBsAg-positive sera after amplification using the polymerase chain reaction. Liver. 1989 Dec;9(6):322–328. doi: 10.1111/j.1600-0676.1989.tb00419.x. [DOI] [PubMed] [Google Scholar]
  15. Weller I. V., Fowler M. J., Monjardino J., Thomas H. C. The detection of HBV-DNA in serum by molecular hybridisation: a more sensitive method for the detection of complete HBV particles. J Med Virol. 1982;9(4):273–280. doi: 10.1002/jmv.1890090405. [DOI] [PubMed] [Google Scholar]