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. 1990 Jun;28(6):1411–1416. doi: 10.1128/jcm.28.6.1411-1416.1990

Detection of hepatitis B virus DNA in serum by polymerase chain reaction amplification and microtiter sandwich hybridization.

G H Keller 1, D P Huang 1, J W Shih 1, M M Manak 1
PMCID: PMC267941  PMID: 2199501

Abstract

We have developed a microtiter sandwich hybridization assay for the detection of polymerase chain reaction (PCR)-amplified hepatitis B virus (HBV) sequences. This assay utilizes an enzyme-linked immunosorbent assay-like format in which cloned DNA containing a sequence complementary to half of one PCR product strand is immobilized in microtiter wells. A biotin-labeled DNA sequence complementary to the other portion of the same PCR product strand is used as the probe. The DNAs from 69 hepatitis B surface antigen-positive serum samples and 16 antigen-negative control samples were amplified by the PCR procedure, and the product was detected by Southern and sandwich hybridization. Both detection procedures were capable of detecting as few as five copies of HBV DNA. Compared with Southern hybridization, the sandwich hybridization assay exhibited a sensitivity of 100% and a specificity of 95% for the detection of amplified HBV sequences. Unlike Southern hybridization, however, the sandwich hybridization assay employs a nonradioactive probe and allows easy handling of large numbers of samples. DNA was detected in 74% of the antigen-positive samples. All of the antigen-negative samples (healthy blood donors) were negative for HBV DNA by both procedures.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Aach R. D., Kahn R. A. Post-transfusion hepatitis: current perspectives. Ann Intern Med. 1980 Apr;92(4):539–546. doi: 10.7326/0003-4819-92-4-539. [DOI] [PubMed] [Google Scholar]
  2. Bréchot C., Degos F., Lugassy C., Thiers V., Zafrani S., Franco D., Bismuth H., Trépo C., Benhamou J. P., Wands J. Hepatitis B virus DNA in patients with chronic liver disease and negative tests for hepatitis B surface antigen. N Engl J Med. 1985 Jan 31;312(5):270–276. doi: 10.1056/NEJM198501313120503. [DOI] [PubMed] [Google Scholar]
  3. Hindman S. H., Gravelle C. R., Murphy B. L., Bradley D. W., Budge W. R., Maynard J. E. "e" Antigen, Dane particles, and serum DNA polymerase activity in HBsAg carriers. Ann Intern Med. 1976 Oct;85(4):458–460. doi: 10.7326/0003-4819-85-4-458. [DOI] [PubMed] [Google Scholar]
  4. Kaneko S., Feinstone S. M., Miller R. H. Rapid and sensitive method for the detection of serum hepatitis B virus DNA using the polymerase chain reaction technique. J Clin Microbiol. 1989 Sep;27(9):1930–1933. doi: 10.1128/jcm.27.9.1930-1933.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. 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]
  6. Keller G. H., Cumming C. U., Huang D. P., Manak M. M., Ting R. A chemical method for introducing haptens onto DNA probes. Anal Biochem. 1988 May 1;170(2):441–450. doi: 10.1016/0003-2697(88)90656-2. [DOI] [PubMed] [Google Scholar]
  7. Keller G. H., Huang D. P., Manak M. M. A sensitive nonisotopic hybridization assay for HIV-1 DNA. Anal Biochem. 1989 Feb 15;177(1):27–32. doi: 10.1016/0003-2697(89)90007-9. [DOI] [PubMed] [Google Scholar]
  8. Kwok S., Higuchi R. Avoiding false positives with PCR. Nature. 1989 May 18;339(6221):237–238. doi: 10.1038/339237a0. [DOI] [PubMed] [Google Scholar]
  9. Lawn R. M., Efstratiadis A., O'Connell C., Maniatis T. The nucleotide sequence of the human beta-globin gene. Cell. 1980 Oct;21(3):647–651. doi: 10.1016/0092-8674(80)90428-6. [DOI] [PubMed] [Google Scholar]
  10. Moriarty A. M., Hoyer B. H., Shih J. W., Gerin J. L., Hamer D. H. Expression of the hepatitis B virus surface antigen gene in cell culture by using a simian virus 40 vector. Proc Natl Acad Sci U S A. 1981 Apr;78(4):2606–2610. doi: 10.1073/pnas.78.4.2606. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Nagata Y., Yokota H., Kosuda O., Yokoo K., Takemura K., Kikuchi T. Quantification of picogram levels of specific DNA immobilized in microtiter wells. FEBS Lett. 1985 Apr 22;183(2):379–382. doi: 10.1016/0014-5793(85)80814-0. [DOI] [PubMed] [Google Scholar]
  12. Nalpas B., Berthelot P., Thiers V., Duhamel G., Courouce A. M., Tiollais P., Brechot C. Hepatitis B virus multiplication in the absence of usual serological markers. A study of 146 chronic alcoholics. J Hepatol. 1985;1(2):89–97. doi: 10.1016/s0168-8278(85)80758-3. [DOI] [PubMed] [Google Scholar]
  13. Ono Y., Onda H., Sasada R., Igarashi K., Sugino Y., Nishioka K. The complete nucleotide sequences of the cloned hepatitis B virus DNA; subtype adr and adw. Nucleic Acids Res. 1983 Mar 25;11(6):1747–1757. doi: 10.1093/nar/11.6.1747. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. 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]
  15. Shafritz D. A., Lieberman H. M., Isselbacher K. J., Wands J. R. Monoclonal radioimmunoassays for hepatitis B surface antigen: demonstration of hepatitis B virus DNA or related sequences in serum and viral epitopes in immune complexes. Proc Natl Acad Sci U S A. 1982 Sep;79(18):5675–5679. doi: 10.1073/pnas.79.18.5675. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Sun C. F., Pao C. C., Wu S. Y., Liaw Y. F. Screening for hepatitis B virus in healthy blood donors by molecular DNA hybridization analysis. J Clin Microbiol. 1988 Sep;26(9):1848–1852. doi: 10.1128/jcm.26.9.1848-1852.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Takahashi K., Imai M., Tsuda F., Takahashi T., Miyakawa Y. Association of dane particles with e antigen in the serum of asymptomatic carriers of hepatitis B surface antigen. J Immunol. 1976 Jul;117(1):102–105. [PubMed] [Google Scholar]
  18. Wu J. S., Chen C. H., Chiang Y. H., Lee Y. C., Lee M. H., Ko Y. C., Hu H. T. Hepatitis B virus infection in Taiwan with reference to anti-HBc versus HBsAg and anti-HBs. Taiwan Yi Xue Hui Za Zhi. 1980 Sep;79(9):760–767. [PubMed] [Google Scholar]

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