Skip to main content
NCBI home page
Proceedings of the National Academy of Sciences of the United States of America logoLink to Proceedings of the National Academy of Sciences of the United States of America
. 1992 Jan 1;89(1):187–191. doi: 10.1073/pnas.89.1.187

Importance of primer selection for the detection of hepatitis C virus RNA with the polymerase chain reaction assay.

J Bukh 1, R H Purcell 1, R H Miller 1
PMCID: PMC48201  PMID: 1309604

Abstract

We compared four primer sets from conserved regions of the hepatitis C virus (HCV) genome for their ability to detect HCV RNA in a "nested" cDNA polymerase chain reaction assay on sera from 114 anti-HCV antibody-positive individuals from around the world. The different primer sets had equivalent sensitivity, detecting less than 1 chimpanzee ID50 (dose that infects 50%) when tested against reference strain H of HCV. We tested equal amounts of RNA extracted from the serum of each individual with the four primer sets. The set derived from two highly conserved domains within the 5' noncoding (NC) region of the HCV genome, which also share significant similarity with Pestivirus 5' NC sequences, was the most effective at detecting HCV RNA. All samples positive for HCV RNA with any other primer set were also positive with the primer set from the 5' NC region, and the latter was at least 3 times more likely to detect HCV infection than a primer set from within the nonstructural protein 3-like gene region (P less than 0.001). We had no false positive results in greater than 500 negative controls interspersed among the test samples. The 5' NC region primer set detected HCV-specific RNA, verified by high-stringency Southern blot hybridization and DNA sequencing, in 100% of 15 acute and 33 chronic non-A, non-B hepatitis patients from the United States, Europe, and Asia and 10 hepatocellular carcinoma patients from Africa and Asia that tested negative for the hepatitis B virus-encoded surface antigen. In conclusion, use of an appropriate primer set is crucial for detecting HCV RNA in the serum of infected individuals.

Full text

PDF
187

Selected References

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

  1. Alberti A. Diagnosis of hepatitis C. Facts and perspectives. J Hepatol. 1991 May;12(3):279–282. doi: 10.1016/0168-8278(91)90827-x. [DOI] [PubMed] [Google Scholar]
  2. Bachmann B., Lüke W., Hunsmann G. Improvement of PCR amplified DNA sequencing with the aid of detergents. Nucleic Acids Res. 1990 Mar 11;18(5):1309–1309. doi: 10.1093/nar/18.5.1309. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. 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]
  4. 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]
  5. Cristiano K., Di Bisceglie A. M., Hoofnagle J. H., Feinstone S. M. Hepatitis C viral RNA in serum of patients with chronic non-A, non-B hepatitis: detection by the polymerase chain reaction using multiple primer sets. Hepatology. 1991 Jul;14(1):51–55. doi: 10.1002/hep.1840140109. [DOI] [PubMed] [Google Scholar]
  6. 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]
  7. Farci P., Alter H. J., Wong D., Miller R. H., Shih J. W., Jett B., Purcell R. H. A long-term study of hepatitis C virus replication in non-A, non-B hepatitis. N Engl J Med. 1991 Jul 11;325(2):98–104. doi: 10.1056/NEJM199107113250205. [DOI] [PubMed] [Google Scholar]
  8. Garson J. A., Ring C., Tuke P., Tedder R. S. Enhanced detection by PCR of hepatitis C virus RNA. Lancet. 1990 Oct 6;336(8719):878–879. doi: 10.1016/0140-6736(90)92384-t. [DOI] [PubMed] [Google Scholar]
  9. Garson J. A., Tedder R. S., Briggs M., Tuke P., Glazebrook J. A., Trute A., Parker D., Barbara J. A., Contreras M., Aloysius S. Detection of hepatitis C viral sequences in blood donations by "nested" polymerase chain reaction and prediction of infectivity. Lancet. 1990 Jun 16;335(8703):1419–1422. doi: 10.1016/0140-6736(90)91446-h. [DOI] [PubMed] [Google Scholar]
  10. Garson J. A., Tuke P. W., Makris M., Briggs M., Machin S. J., Preston F. E., Tedder R. S. Demonstration of viraemia patterns in haemophiliacs treated with hepatitis-C-virus-contaminated factor VIII concentrates. Lancet. 1990 Oct 27;336(8722):1022–1025. doi: 10.1016/0140-6736(90)92487-3. [DOI] [PubMed] [Google Scholar]
  11. Han J. H., Shyamala V., Richman K. H., Brauer M. J., Irvine B., Urdea M. S., Tekamp-Olson P., Kuo G., Choo Q. L., Houghton M. Characterization of the terminal regions of hepatitis C viral RNA: identification of conserved sequences in the 5' untranslated region and poly(A) tails at the 3' end. Proc Natl Acad Sci U S A. 1991 Mar 1;88(5):1711–1715. doi: 10.1073/pnas.88.5.1711. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Hijikata M., Kato N., Mori S., Ootsuyama Y., Nakagawa M., Sugimura T., Ohkoshi S., Kojima H., Meguro T., Taki M. Frequent detection of hepatitis C virus US strain in Japanese hemophiliacs. Jpn J Cancer Res. 1990 Dec;81(12):1195–1197. doi: 10.1111/j.1349-7006.1990.tb02676.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Hijikata M., Kato N., Ootsuyama Y., Nakagawa M., Ohkoshi S., Shimotohno K. Hypervariable regions in the putative glycoprotein of hepatitis C virus. Biochem Biophys Res Commun. 1991 Feb 28;175(1):220–228. doi: 10.1016/s0006-291x(05)81223-9. [DOI] [PubMed] [Google Scholar]
  14. Houghton M., Weiner A., Han J., Kuo G., Choo Q. L. Molecular biology of the hepatitis C viruses: implications for diagnosis, development and control of viral disease. Hepatology. 1991 Aug;14(2):381–388. [PubMed] [Google Scholar]
  15. Kaneko S., Unoura M., Kobayashi K., Kuno K., Murakami S., Hattori N. Detection of serum hepatitis C virus RNA. Lancet. 1990 Apr 21;335(8695):976–976. doi: 10.1016/0140-6736(90)91042-9. [DOI] [PubMed] [Google Scholar]
  16. Kato N., Hijikata M., Ootsuyama Y., Nakagawa M., Ohkoshi S., Shimotohno K. Sequence diversity of hepatitis C viral genomes. Mol Biol Med. 1990 Dec;7(6):495–501. [PubMed] [Google Scholar]
  17. Kato N., Hijikata M., Ootsuyama Y., Nakagawa M., Ohkoshi S., Sugimura T., Shimotohno K. Molecular cloning of the human hepatitis C virus genome from Japanese patients with non-A, non-B hepatitis. Proc Natl Acad Sci U S A. 1990 Dec;87(24):9524–9528. doi: 10.1073/pnas.87.24.9524. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Kato N., Yokosuka O., Omata M., Hosoda K., Ohto M. Detection of hepatitis C virus ribonucleic acid in the serum by amplification with polymerase chain reaction. J Clin Invest. 1990 Nov;86(5):1764–1767. doi: 10.1172/JCI114903. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Kubo Y., Takeuchi K., Boonmar S., Katayama T., Choo Q. L., Kuo G., Weiner A. J., Bradley D. W., Houghton M., Saito I. A cDNA fragment of hepatitis C virus isolated from an implicated donor of post-transfusion non-A, non-B hepatitis in Japan. Nucleic Acids Res. 1989 Dec 25;17(24):10367–10372. doi: 10.1093/nar/17.24.10367. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Kuo G., Choo Q. L., Alter H. J., Gitnick G. L., Redeker A. G., Purcell R. H., Miyamura T., Dienstag J. L., Alter M. J., Stevens C. E. An assay for circulating antibodies to a major etiologic virus of human non-A, non-B hepatitis. Science. 1989 Apr 21;244(4902):362–364. doi: 10.1126/science.2496467. [DOI] [PubMed] [Google Scholar]
  21. 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]
  22. McFarlane I. G., Smith H. M., Johnson P. J., Bray G. P., Vergani D., Williams R. Hepatitis C virus antibodies in chronic active hepatitis: pathogenetic factor or false-positive result? Lancet. 1990 Mar 31;335(8692):754–757. doi: 10.1016/0140-6736(90)90870-b. [DOI] [PubMed] [Google Scholar]
  23. Miller R. H., Lee S. C., Liaw Y. F., Robinson W. S. Hepatitis B viral DNA in infected human liver and in hepatocellular carcinoma. J Infect Dis. 1985 Jun;151(6):1081–1092. doi: 10.1093/infdis/151.6.1081. [DOI] [PubMed] [Google Scholar]
  24. Miller R. H., Purcell R. H. Hepatitis C virus shares amino acid sequence similarity with pestiviruses and flaviviruses as well as members of two plant virus supergroups. Proc Natl Acad Sci U S A. 1990 Mar;87(6):2057–2061. doi: 10.1073/pnas.87.6.2057. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. 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]
  26. Ohkoshi S., Kato N., Kinoshita T., Hijikata M., Ohtsuyama Y., Okazaki N., Ohkura H., Hirohashi S., Honma A., Ozaki T. Detection of hepatitis C virus RNA in sera and liver tissues of non-A, non-B hepatitis patients using the polymerase chain reaction. Jpn J Cancer Res. 1990 Sep;81(9):862–865. doi: 10.1111/j.1349-7006.1990.tb02658.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Okamoto H., Okada S., Sugiyama Y., Tanaka T., Sugai Y., Akahane Y., Machida A., Mishiro S., Yoshizawa H., Miyakawa Y. Detection of hepatitis C virus RNA by a two-stage polymerase chain reaction with two pairs of primers deduced from the 5'-noncoding region. Jpn J Exp Med. 1990 Aug;60(4):215–222. [PubMed] [Google Scholar]
  28. Okamoto H., Okada S., Sugiyama Y., Yotsumoto S., Tanaka T., Yoshizawa H., Tsuda F., Miyakawa Y., Mayumi M. The 5'-terminal sequence of the hepatitis C virus genome. Jpn J Exp Med. 1990 Jun;60(3):167–177. [PubMed] [Google Scholar]
  29. Shibata M., Morishima T., Kudo T., Maki T., Maki S., Nagai Y. Serum hepatitis C virus sequences in posttransfusion non-A, non-B hepatitis. Blood. 1991 Mar 15;77(6):1157–1160. [PubMed] [Google Scholar]
  30. Simmonds P., Zhang L. Q., Watson H. G., Rebus S., Ferguson E. D., Balfe P., Leadbetter G. H., Yap P. L., Peutherer J. F., Ludlam C. A. Hepatitis C quantification and sequencing in blood products, haemophiliacs, and drug users. Lancet. 1990 Dec 15;336(8729):1469–1472. doi: 10.1016/0140-6736(90)93179-s. [DOI] [PubMed] [Google Scholar]
  31. Takamizawa A., Mori C., Fuke I., Manabe S., Murakami S., Fujita J., Onishi E., Andoh T., Yoshida I., Okayama H. Structure and organization of the hepatitis C virus genome isolated from human carriers. J Virol. 1991 Mar;65(3):1105–1113. doi: 10.1128/jvi.65.3.1105-1113.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Takeuchi K., Boonmar S., Kubo Y., Katayama T., Harada H., Ohbayashi A., Choo Q. L., Kuo G., Houghton M., Saito I. Hepatitis C viral cDNA clones isolated from a healthy carrier donor implicated in post-transfusion non-A, non-B hepatitis. Gene. 1990 Jul 16;91(2):287–291. doi: 10.1016/0378-1119(90)90102-w. [DOI] [PubMed] [Google Scholar]
  33. Takeuchi K., Kubo Y., Boonmar S., Watanabe Y., Katayama T., Choo Q. L., Kuo G., Houghton M., Saito I., Miyamura T. The putative nucleocapsid and envelope protein genes of hepatitis C virus determined by comparison of the nucleotide sequences of two isolates derived from an experimentally infected chimpanzee and healthy human carriers. J Gen Virol. 1990 Dec;71(Pt 12):3027–3033. doi: 10.1099/0022-1317-71-12-3027. [DOI] [PubMed] [Google Scholar]
  34. Tedder R. S., Gilson R. J., Briggs M., Loveday C., Cameron C. H., Garson J. A., Kelly G. E., Weller I. V. Hepatitis C virus: evidence for sexual transmission. BMJ. 1991 Jun 1;302(6788):1299–1302. doi: 10.1136/bmj.302.6788.1299. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Ulrich P. P., Romeo J. M., Lane P. K., Kelly I., Daniel L. J., Vyas G. N. Detection, semiquantitation, and genetic variation in hepatitis C virus sequences amplified from the plasma of blood donors with elevated alanine aminotransferase. J Clin Invest. 1990 Nov;86(5):1609–1614. doi: 10.1172/JCI114882. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Van der Poel C. L., Cuypers H. T., Reesink H. W., Weiner A. J., Quan S., Di Nello R., Van Boven J. J., Winkel I., Mulder-Folkerts D., Exel-Oehlers P. J. Confirmation of hepatitis C virus infection by new four-antigen recombinant immunoblot assay. Lancet. 1991 Feb 9;337(8737):317–319. doi: 10.1016/0140-6736(91)90942-i. [DOI] [PubMed] [Google Scholar]
  37. Weiner A. J., Brauer M. J., Rosenblatt J., Richman K. H., Tung J., Crawford K., Bonino F., Saracco G., Choo Q. L., Houghton M. Variable and hypervariable domains are found in the regions of HCV corresponding to the flavivirus envelope and NS1 proteins and the pestivirus envelope glycoproteins. Virology. 1991 Feb;180(2):842–848. doi: 10.1016/0042-6822(91)90104-j. [DOI] [PubMed] [Google Scholar]
  38. Weiner A. J., Kuo G., Bradley D. W., Bonino F., Saracco G., Lee C., Rosenblatt J., Choo Q. L., Houghton M. Detection of hepatitis C viral sequences in non-A, non-B hepatitis. Lancet. 1990 Jan 6;335(8680):1–3. doi: 10.1016/0140-6736(90)90134-q. [DOI] [PubMed] [Google Scholar]
  39. Wong D. C., Diwan A. R., Rosen L., Gerin J. L., Johnson R. G., Polito A., Purcell R. H. Non-specificity of anti-HCV test for seroepidemiological analysis. Lancet. 1990 Sep 22;336(8717):750–751. doi: 10.1016/0140-6736(90)92245-d. [DOI] [PubMed] [Google Scholar]
  40. Yolken R., Dubovi E., Leister F., Reid R., Almeido-Hill J., Santosham M. Infantile gastroenteritis associated with excretion of pestivirus antigens. Lancet. 1989 Mar 11;1(8637):517–520. doi: 10.1016/s0140-6736(89)90066-4. [DOI] [PubMed] [Google Scholar]
  41. Zonaro A., Puoti M., Fiordalisi G., Mantero G., Castelnuovo F., Primi D., Cariani E. Detection of serum hepatitis C virus RNA in acute non-A, non-B hepatitis. J Infect Dis. 1991 Apr;163(4):923–924. doi: 10.1093/infdis/163.4.923a. [DOI] [PubMed] [Google Scholar]

Articles from Proceedings of the National Academy of Sciences of the United States of America are provided here courtesy of National Academy of Sciences

RESOURCES