Skip to main content
PMC home page
Journal of Clinical Microbiology logoLink to Journal of Clinical Microbiology
. 1994 Aug;32(8):1887–1893. doi: 10.1128/jcm.32.8.1887-1893.1994

Automated quantitative determination of hepatitis C virus viremia by reverse transcription-PCR.

N C Besnard 1, P M Andre 1
PMCID: PMC263897  PMID: 7989537

Abstract

An automated reverse transcription-PCR was developed for the quantitative detection of hepatitis C virus. The quantitation is based on the coamplification and labelling with digoxigenin-dUTP during PCR of two similar templates, the viral genome and a modified RNA which acts as a mimic target. Known amounts of the mimic RNA sequence were introduced into the clinical samples. The automated quantitation of the two coamplified and labelled products depends on the use of two biotinylated caputre probes which are complementary, respectively, to a deleted sequence and to an inserted sequence introduced by site-directed mutagenesis in a wild viral cloned cDNA. This method proved to be simple, reproducible, and useful for quantitate hepatitis C virus viremia in chronically infected patients. This easy-to-perform, automated assay could also be used for the accurate determination of human immunodeficiency virus viremia or other RNA molecules.

Full text

PDF
1890

Selected References

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

  1. Bouaboula M., Legoux P., Pességué B., Delpech B., Dumont X., Piechaczyk M., Casellas P., Shire D. Standardization of mRNA titration using a polymerase chain reaction method involving co-amplification with a multispecific internal control. J Biol Chem. 1992 Oct 25;267(30):21830–21838. [PubMed] [Google Scholar]
  2. Brillanti S., Garson J. A., Tuke P. W., Ring C., Briggs M., Masci C., Miglioli M., Barbara L., Tedder R. S. Effect of alpha-interferon therapy on hepatitis C viraemia in community-acquired chronic non-A, non-B hepatitis: a quantitative polymerase chain reaction study. J Med Virol. 1991 Jun;34(2):136–141. doi: 10.1002/jmv.1890340213. [DOI] [PubMed] [Google Scholar]
  3. Bukh J., Purcell R. H., Miller R. H. Sequence analysis of the 5' noncoding region of hepatitis C virus. Proc Natl Acad Sci U S A. 1992 Jun 1;89(11):4942–4946. doi: 10.1073/pnas.89.11.4942. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Chen P. J., Lin M. H., Tai K. F., Liu P. C., Lin C. J., Chen D. S. The Taiwanese hepatitis C virus genome: sequence determination and mapping the 5' termini of viral genomic and antigenomic RNA. Virology. 1992 May;188(1):102–113. doi: 10.1016/0042-6822(92)90739-c. [DOI] [PubMed] [Google Scholar]
  5. Chomczynski P., Sacchi N. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem. 1987 Apr;162(1):156–159. doi: 10.1006/abio.1987.9999. [DOI] [PubMed] [Google Scholar]
  6. Di Bisceglie A. M., Martin P., Kassianides C., Lisker-Melman M., Murray L., Waggoner J., Goodman Z., Banks S. M., Hoofnagle J. H. Recombinant interferon alfa therapy for chronic hepatitis C. A randomized, double-blind, placebo-controlled trial. N Engl J Med. 1989 Nov 30;321(22):1506–1510. doi: 10.1056/NEJM198911303212204. [DOI] [PubMed] [Google Scholar]
  7. Gilliland G., Perrin S., Blanchard K., Bunn H. F. Analysis of cytokine mRNA and DNA: detection and quantitation by competitive polymerase chain reaction. Proc Natl Acad Sci U S A. 1990 Apr;87(7):2725–2729. doi: 10.1073/pnas.87.7.2725. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Griffais R., André P. M., Thibon M. K-tuple frequency in the human genome and polymerase chain reaction. Nucleic Acids Res. 1991 Jul 25;19(14):3887–3891. doi: 10.1093/nar/19.14.3887. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Griffais R., André P. M., Thibon M. Synthesis of digoxigenin-labelled DNA probe by polymerase chain reaction: application to Epstein-Barr virus and Chlamydia trachomatis. Res Virol. 1990 May-Jun;141(3):331–335. doi: 10.1016/0923-2516(90)90004-3. [DOI] [PubMed] [Google Scholar]
  10. Hagiwara H., Hayashi N., Mita E., Takehara T., Kasahara A., Fusamoto H., Kamada T. Quantitative analysis of hepatitis C virus RNA in serum during interferon alfa therapy. Gastroenterology. 1993 Mar;104(3):877–883. doi: 10.1016/0016-5085(93)91025-d. [DOI] [PubMed] [Google Scholar]
  11. Hoofnagle J. H., Mullen K. D., Jones D. B., Rustgi V., Di Bisceglie A., Peters M., Waggoner J. G., Park Y., Jones E. A. Treatment of chronic non-A,non-B hepatitis with recombinant human alpha interferon. A preliminary report. N Engl J Med. 1986 Dec 18;315(25):1575–1578. doi: 10.1056/NEJM198612183152503. [DOI] [PubMed] [Google Scholar]
  12. Kaneko S., Murakami S., Unoura M., Kobayashi K. Quantitation of hepatitis C virus RNA by competitive polymerase chain reaction. J Med Virol. 1992 Aug;37(4):278–282. doi: 10.1002/jmv.1890370408. [DOI] [PubMed] [Google Scholar]
  13. Kleter G. E., van Doorn L. J., Brouwer J. T., Schalm S. W., Heijtink R. A., Quint W. G. Sequence analysis of the 5' untranslated region in isolates of at least four genotypes of hepatitis C virus in The Netherlands. J Clin Microbiol. 1994 Feb;32(2):306–310. doi: 10.1128/jcm.32.2.306-310.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Lau J. Y., Davis G. L., Kniffen J., Qian K. P., Urdea M. S., Chan C. S., Mizokami M., Neuwald P. D., Wilber J. C. Significance of serum hepatitis C virus RNA levels in chronic hepatitis C. Lancet. 1993 Jun 12;341(8859):1501–1504. doi: 10.1016/0140-6736(93)90635-t. [DOI] [PubMed] [Google Scholar]
  15. Magrin S., Craxì A., Fabiano C., Fiorentino G., Marino L., Almasio P., Pinzello G. B., Palazzo U., Vitale M., Maggio A. Serum hepatitis C virus (HCV)-RNA and response to alpha-interferon in anti-HCV positive chronic hepatitis. J Med Virol. 1992 Nov;38(3):200–206. doi: 10.1002/jmv.1890380309. [DOI] [PubMed] [Google Scholar]
  16. Ou C. Y., McDonough S. H., Cabanas D., Ryder T. B., Harper M., Moore J., Schochetman G. Rapid and quantitative detection of enzymatically amplified HIV-1 DNA using chemiluminescent oligonucleotide probes. AIDS Res Hum Retroviruses. 1990 Nov;6(11):1323–1329. doi: 10.1089/aid.1990.6.1323. [DOI] [PubMed] [Google Scholar]
  17. Piatak M., Jr, Luk K. C., Williams B., Lifson J. D. Quantitative competitive polymerase chain reaction for accurate quantitation of HIV DNA and RNA species. Biotechniques. 1993 Jan;14(1):70–81. [PubMed] [Google Scholar]
  18. Simmonds P., Balfe P., Peutherer J. F., Ludlam C. A., Bishop J. O., Brown A. J. Human immunodeficiency virus-infected individuals contain provirus in small numbers of peripheral mononuclear cells and at low copy numbers. J Virol. 1990 Feb;64(2):864–872. doi: 10.1128/jvi.64.2.864-872.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Wang A. M., Doyle M. V., Mark D. F. Quantitation of mRNA by the polymerase chain reaction. Proc Natl Acad Sci U S A. 1989 Dec;86(24):9717–9721. doi: 10.1073/pnas.86.24.9717. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Journal of Clinical Microbiology are provided here courtesy of American Society for Microbiology (ASM)

RESOURCES