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. 1996 Apr;34(4):901–907. doi: 10.1128/jcm.34.4.901-907.1996

Accurate quantification of hepatitis C virus (HCV) RNA from all HCV genotypes by using branched-DNA technology.

J Detmer 1, R Lagier 1, J Flynn 1, C Zayati 1, J Kolberg 1, M Collins 1, M Urdea 1, R Sánchez-Pescador 1
PMCID: PMC228914  PMID: 8815105

Abstract

In studies monitoring disease progression and therapeutic response, it is essential that the method used for hepatitis C virus (HCV) quantification not be influenced by genotypic variability. The branched DNA assay provides a reliable method for the quantification of HCV RNA. A modified set of oligonucleotide probes for the branched DNA assay was developed to enhance the efficiency of binding to genotypic variants of HCV. The improved branched DNA assay (HCV RNA 2.0) yielded highly reproducible quantification of hepatitis C virus RNA and displayed a nearly 600-fold dynamic range in quantification up to 120 Meq of HCV RNA per ml. The quantification limit was set at 0.2 Meg of HCV RNA per ml to ensure a specificity of > or = 95%. With this lowered quantification limit and the enhanced hybridization of the probes, the HCV RNA 2.0 assay exhibited a high level of sensitivity (96%) and was virtually unaffected by the genotypic variability of HCV. The HCV RNA 2.0 assay may be a useful tool for following HCV RNA levels throughout the course of disease, selecting patients for therapy, and evaluating therapeutic response.

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Selected References

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  1. Alter H. J., Sanchez-Pescador R., Urdea M. S., Wilber J. C., Lagier R. J., Di Bisceglie A. M., Shih J. W., Neuwald P. D. Evaluation of branched DNA signal amplification for the detection of hepatitis C virus RNA. J Viral Hepat. 1995;2(3):121–132. doi: 10.1111/j.1365-2893.1995.tb00017.x. [DOI] [PubMed] [Google Scholar]
  2. Bresters D., Cuypers H. T., Reesink H. W., Mauser-Bunschoten E. P., van den Berg H. M., Schaasberg W. P., Wilber J. C., Urdea M. S., Neuwald P., Lelie P. N. Comparison of quantitative cDNA-PCR with the branched DNA hybridization assay for monitoring plasma hepatitis C virus RNA levels in haemophilia patients participating in a controlled interferon trial. J Med Virol. 1994 Jul;43(3):262–268. doi: 10.1002/jmv.1890430313. [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. Chan C. Y., Lee S. D., Hwang S. J., Lu R. H., Lu C. L., Lo K. J. Quantitative branched DNA assay and genotyping for hepatitis C virus RNA in Chinese patients with acute and chronic hepatitis C. J Infect Dis. 1995 Feb;171(2):443–446. doi: 10.1093/infdis/171.2.443. [DOI] [PubMed] [Google Scholar]
  5. Chazouilleres O., Kim M., Combs C., Ferrell L., Bacchetti P., Roberts J., Ascher N. L., Neuwald P., Wilber J., Urdea M. Quantitation of hepatitis C virus RNA in liver transplant recipients. Gastroenterology. 1994 Apr;106(4):994–999. doi: 10.1016/0016-5085(94)90759-5. [DOI] [PubMed] [Google Scholar]
  6. Chemello L., Alberti A., Rose K., Simmonds P. Hepatitis C serotype and response to interferon therapy. N Engl J Med. 1994 Jan 13;330(2):143–143. doi: 10.1056/NEJM199401133300215. [DOI] [PubMed] [Google Scholar]
  7. Choo Q. L., Kuo G., Weiner A. J., Overby L. R., Bradley D. W., Houghton M. Isolation of a cDNA clone derived from a blood-borne non-A, non-B viral hepatitis genome. Science. 1989 Apr 21;244(4902):359–362. doi: 10.1126/science.2523562. [DOI] [PubMed] [Google Scholar]
  8. Choo Q. L., Richman K. H., Han J. H., Berger K., Lee C., Dong C., Gallegos C., Coit D., Medina-Selby R., Barr P. J. Genetic organization and diversity of the hepatitis C virus. Proc Natl Acad Sci U S A. 1991 Mar 15;88(6):2451–2455. doi: 10.1073/pnas.88.6.2451. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Choo Q. L., Weiner A. J., Overby L. R., Kuo G., Houghton M., Bradley D. W. Hepatitis C virus: the major causative agent of viral non-A, non-B hepatitis. Br Med Bull. 1990 Apr;46(2):423–441. doi: 10.1093/oxfordjournals.bmb.a072408. [DOI] [PubMed] [Google Scholar]
  10. Coelho-Little E., Jeffers L. J., Bartholomew M., Reddy K. R., Schiff E. R., Dailey P. J. Correlation of HCV-RNA levels in serum and liver of patients with chronic hepatitis C. J Hepatol. 1995 Apr;22(4):508–508. doi: 10.1016/0168-8278(95)80118-9. [DOI] [PubMed] [Google Scholar]
  11. Collins M. L., Zayati C., Detmer J. J., Daly B., Kolberg J. A., Cha T. A., Irvine B. D., Tucker J., Urdea M. S. Preparation and characterization of RNA standards for use in quantitative branched DNA hybridization assays. Anal Biochem. 1995 Mar 20;226(1):120–129. doi: 10.1006/abio.1995.1199. [DOI] [PubMed] [Google Scholar]
  12. Daly J. A., Ertingshausen G. Direct method for determining inorganic phosphate in serum with the "CentrifiChem". Clin Chem. 1972 Mar;18(3):263–265. [PubMed] [Google Scholar]
  13. Davis G. L., Lau J. Y., Urdea M. S., Neuwald P. D., Wilber J. C., Lindsay K., Perrillo R. P., Albrecht J. Quantitative detection of hepatitis C virus RNA with a solid-phase signal amplification method: definition of optimal conditions for specimen collection and clinical application in interferon-treated patients. Hepatology. 1994 Jun;19(6):1337–1341. [PubMed] [Google Scholar]
  14. Davis G. L. Prediction of response to interferon treatment of chronic hepatitis C. J Hepatol. 1994 Jul;21(1):1–3. doi: 10.1016/s0168-8278(94)80128-2. [DOI] [PubMed] [Google Scholar]
  15. Diodati G., Bonetti P., Noventa F., Casarin C., Rugge M., Scaccabarozzi S., Tagger A., Pollice L., Tremolada F., Davite C. Treatment of chronic hepatitis C with recombinant human interferon-alpha 2a: results of a randomized controlled clinical trial. Hepatology. 1994 Jan;19(1):1–5. [PubMed] [Google Scholar]
  16. Enomoto N., Takada A., Nakao T., Date T. There are two major types of hepatitis C virus in Japan. Biochem Biophys Res Commun. 1990 Aug 16;170(3):1021–1025. doi: 10.1016/0006-291x(90)90494-8. [DOI] [PubMed] [Google Scholar]
  17. Féray C., Gigou M., Samuel D., Paradis V., Wilber J., David M. F., Urdea M., Reynes M., Bréchot C., Bismuth H. The course of hepatitis C virus infection after liver transplantation. Hepatology. 1994 Nov;20(5):1137–1143. doi: 10.1002/hep.1840200506. [DOI] [PubMed] [Google Scholar]
  18. Gordon S. C., Kodali V. P., Silverman A. L., Dmuchowski C. F., Urdea M. S., Chan C. S., Wilber J. C. Levels of hepatitis C virus RNA and liver histology in chronic type C hepatitis. Am J Gastroenterol. 1994 Sep;89(9):1458–1461. [PubMed] [Google Scholar]
  19. Hayashi J., Ohmiya M., Kishihara Y., Tani Y., Kinukawa N., Ikematsu H., Kashiwagi S. A statistical analysis of predictive factors of response to human lymphoblastoid interferon in patients with chronic hepatitis C. Am J Gastroenterol. 1994 Dec;89(12):2151–2156. [PubMed] [Google Scholar]
  20. Horn T., Urdea M. S. Forks and combs and DNA: the synthesis of branched oligodeoxyribonucleotides. Nucleic Acids Res. 1989 Sep 12;17(17):6959–6967. doi: 10.1093/nar/17.17.6959. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Kanai K., Kako M., Okamoto H. HCV genotypes in chronic hepatitis C and response to interferon. Lancet. 1992 Jun 20;339(8808):1543–1543. doi: 10.1016/0140-6736(92)91311-u. [DOI] [PubMed] [Google Scholar]
  22. 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]
  23. Lau J. Y., Mizokami M., Kolberg J. A., Davis G. L., Prescott L. E., Ohno T., Perrillo R. P., Lindsay K. L., Gish R. G., Qian K. P. Application of six hepatitis C virus genotyping systems to sera from chronic hepatitis C patients in the United States. J Infect Dis. 1995 Feb;171(2):281–289. doi: 10.1093/infdis/171.2.281. [DOI] [PubMed] [Google Scholar]
  24. Lau J. Y., Simmonds P., Urdea M. S. Implications of variations of "conserved" regions of hepatitis C virus genome. Lancet. 1995 Aug 12;346(8972):425–426. doi: 10.1016/s0140-6736(95)92786-7. [DOI] [PubMed] [Google Scholar]
  25. Lin L. I. A concordance correlation coefficient to evaluate reproducibility. Biometrics. 1989 Mar;45(1):255–268. [PubMed] [Google Scholar]
  26. Magrin S., Craxi A., Fabiano C., Simonetti R. G., Fiorentino G., Marino L., Diquattro O., Di Marco V., Loiacono O., Volpes R. Hepatitis C viremia in chronic liver disease: relationship to interferon-alpha or corticosteroid treatment. Hepatology. 1994 Feb;19(2):273–279. [PubMed] [Google Scholar]
  27. Mahaney K., Tedeschi V., Maertens G., Di Bisceglie A. M., Vergalla J., Hoofnagle J. H., Sallie R. Genotypic analysis of hepatitis C virus in American patients. Hepatology. 1994 Dec;20(6):1405–1411. doi: 10.1002/hep.1840200605. [DOI] [PubMed] [Google Scholar]
  28. Matsumoto A., Tanaka E., Suzuki T., Ogata H., Kiyosawa K. Viral and host factors that contribute to efficacy of interferon-alpha 2a therapy in patients with chronic hepatitis C. Dig Dis Sci. 1994 Jun;39(6):1273–1280. doi: 10.1007/BF02093793. [DOI] [PubMed] [Google Scholar]
  29. Mita E., Hayashi N., Hagiwara H., Ueda K., Kanazawa Y., Kasahara A., Fusamoto H., Kamada T. Predicting interferon therapy efficacy from hepatitis C virus genotype and RNA titer. Dig Dis Sci. 1994 May;39(5):977–982. doi: 10.1007/BF02087547. [DOI] [PubMed] [Google Scholar]
  30. Nousbaum J. B., Pol S., Nalpas B., Landais P., Berthelot P., Bréchot C. Hepatitis C virus type 1b (II) infection in France and Italy. Collaborative Study Group. Ann Intern Med. 1995 Feb 1;122(3):161–168. doi: 10.7326/0003-4819-122-3-199502010-00001. [DOI] [PubMed] [Google Scholar]
  31. Ogata N., Alter H. J., Miller R. H., Purcell R. H. Nucleotide sequence and mutation rate of the H strain of hepatitis C virus. Proc Natl Acad Sci U S A. 1991 Apr 15;88(8):3392–3396. doi: 10.1073/pnas.88.8.3392. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Okamoto H., Kojima M., Okada S., Yoshizawa H., Iizuka H., Tanaka T., Muchmore E. E., Peterson D. A., Ito Y., Mishiro S. Genetic drift of hepatitis C virus during an 8.2-year infection in a chimpanzee: variability and stability. Virology. 1992 Oct;190(2):894–899. doi: 10.1016/0042-6822(92)90933-g. [DOI] [PubMed] [Google Scholar]
  33. Orito E., Mizokami M., Nakano T., Terashima H., Nojiri O., Sakakibara K., Mizuno M., Ogino M., Nakamura M., Matsumoto Y. Serum hepatitis C virus RNA level as a predictor of subsequent response to interferon-alpha therapy in Japanese patients with chronic hepatitis C. J Med Virol. 1994 Dec;44(4):410–414. doi: 10.1002/jmv.1890440418. [DOI] [PubMed] [Google Scholar]
  34. Pozzato G., Moretti M., Crocé L. S., Sasso F., Kaneko S., Unoura M., Kobayashi K., Crovatto M., Santini G., Tiribelli C. Interferon therapy in chronic hepatitis C virus: evidence of different outcome with respect to different viral strains. J Med Virol. 1995 Apr;45(4):445–450. doi: 10.1002/jmv.1890450416. [DOI] [PubMed] [Google Scholar]
  35. Running J. A., Urdea M. S. A procedure for productive coupling of synthetic oligonucleotides to polystyrene microtiter wells for hybridization capture. Biotechniques. 1990 Mar;8(3):276–279. [PubMed] [Google Scholar]
  36. Simmonds P., Alberti A., Alter H. J., Bonino F., Bradley D. W., Brechot C., Brouwer J. T., Chan S. W., Chayama K., Chen D. S. A proposed system for the nomenclature of hepatitis C viral genotypes. Hepatology. 1994 May;19(5):1321–1324. [PubMed] [Google Scholar]
  37. Simmonds P., Holmes E. C., Cha T. A., Chan S. W., McOmish F., Irvine B., Beall E., Yap P. L., Kolberg J., Urdea M. S. Classification of hepatitis C virus into six major genotypes and a series of subtypes by phylogenetic analysis of the NS-5 region. J Gen Virol. 1993 Nov;74(Pt 11):2391–2399. doi: 10.1099/0022-1317-74-11-2391. [DOI] [PubMed] [Google Scholar]
  38. Simmonds P. Variability of hepatitis C virus. Hepatology. 1995 Feb;21(2):570–583. doi: 10.1002/hep.1840210243. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Stuyver L., Rossau R., Wyseur A., Duhamel M., Vanderborght B., Van Heuverswyn H., Maertens G. Typing of hepatitis C virus isolates and characterization of new subtypes using a line probe assay. J Gen Virol. 1993 Jun;74(Pt 6):1093–1102. doi: 10.1099/0022-1317-74-6-1093. [DOI] [PubMed] [Google Scholar]
  40. Tokita H., Okamoto H., Tsuda F., Song P., Nakata S., Chosa T., Iizuka H., Mishiro S., Miyakawa Y., Mayumi M. Hepatitis C virus variants from Vietnam are classifiable into the seventh, eighth, and ninth major genetic groups. Proc Natl Acad Sci U S A. 1994 Nov 8;91(23):11022–11026. doi: 10.1073/pnas.91.23.11022. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Tsubota A., Chayama K., Ikeda K., Yasuji A., Koida I., Saitoh S., Hashimoto M., Iwasaki S., Kobayashi M., Hiromitsu K. Factors predictive of response to interferon-alpha therapy in hepatitis C virus infection. Hepatology. 1994 May;19(5):1088–1094. [PubMed] [Google Scholar]
  42. Urdea M. S., Horn T., Fultz T. J., Anderson M., Running J. A., Hamren S., Ahle D., Chang C. A. Branched DNA amplification multimers for the sensitive, direct detection of human hepatitis viruses. Nucleic Acids Symp Ser. 1991;(24):197–200. [PubMed] [Google Scholar]
  43. Urdea M. S., Warner B. D., Running J. A., Stempien M., Clyne J., Horn T. A comparison of non-radioisotopic hybridization assay methods using fluorescent, chemiluminescent and enzyme labeled synthetic oligodeoxyribonucleotide probes. Nucleic Acids Res. 1988 Jun 10;16(11):4937–4956. doi: 10.1093/nar/16.11.4937. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Warshaw M. M., Tinoco I., Jr Optical properties of sixteen dinucleoside phosphates. J Mol Biol. 1966 Sep;20(1):29–38. doi: 10.1016/0022-2836(66)90115-x. [DOI] [PubMed] [Google Scholar]
  45. Wright T. L., Combs C., Kim M., Ferrell L., Bacchetti P., Ascher N., Roberts J., Wilber J., Sheridan P., Urdea M. Interferon-alpha therapy for hepatitis C virus infection after liver transplantation. Hepatology. 1994 Oct;20(4 Pt 1):773–779. doi: 10.1002/hep.1840200402. [DOI] [PubMed] [Google Scholar]
  46. Yamada G., Takatani M., Kishi F., Takahashi M., Doi T., Tsuji T., Shin S., Tanno M., Urdea M. S., Kolberg J. A. Efficacy of interferon alfa therapy in chronic hepatitis C patients depends primarily on hepatitis C virus RNA level. Hepatology. 1995 Nov;22(5):1351–1354. [PubMed] [Google Scholar]
  47. Yoshioka K., Kakumu S., Wakita T., Ishikawa T., Itoh Y., Takayanagi M., Higashi Y., Shibata M., Morishima T. Detection of hepatitis C virus by polymerase chain reaction and response to interferon-alpha therapy: relationship to genotypes of hepatitis C virus. Hepatology. 1992 Aug;16(2):293–299. doi: 10.1002/hep.1840160203. [DOI] [PubMed] [Google Scholar]
  48. van Doorn L. J., Kleter B., Stuyver L., Maertens G., Brouwer H., Schalm S., Heijtink R., Quint W. Analysis of hepatitis C virus genotypes by a line probe assay and correlation with antibody profiles. J Hepatol. 1994 Jul;21(1):122–129. doi: 10.1016/s0168-8278(94)80148-7. [DOI] [PubMed] [Google Scholar]

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