• We are sorry, but NCBI web applications do not support your browser and may not function properly. More information
Logo of jcmPermissionsJournals.ASM.orgJournalJCM ArticleJournal InfoAuthorsReviewers
J Clin Microbiol. Apr 1993; 31(4): 882–886.
PMCID: PMC263581

Detection of hepatitis C virus RNA by a combined reverse transcription-polymerase chain reaction assay.

Abstract

Amplification of RNA by the polymerase chain reaction (PCR) is normally a two-step process requiring separate enzymes and buffer conditions. We describe a combined reverse transcription-PCR (RT-PCR) assay for hepatitis C virus (HCV) RNA amplification in which a single enzyme and buffer condition are used. In this assay, both the RT and PCR steps are carried out with the thermoactive DNA polymerase of Thermus thermophilus. A transcription vector containing HCV sequences has also been constructed to generate quantifiable HCV RNA templates that can be used to optimize reaction conditions and to assess the efficiency of amplification. Amplification from < or = 100 copies of RNA was detected reproducibly by gel electrophoresis. The assay sensitivity was increased to 10 RNA copies by hybridization to a probe. The patterns of viremia in three individuals infected with HCV were examined by amplification of HCV RNA from plasma samples collected serially over a period of 1 year. These results were correlated with the times of seroconversion and the onset of rise in levels of alanine aminotransferase in serum. In all three subjects, HCV RNA was detected prior to seroconversion and the initial rise in levels of alanine aminotransferase in serum. Upon seroconversion, HCV RNA fell to a level below the detection limit of the assay. This pattern of transient viremia appears to be characteristic of acute, resolving HCV infections. The combined RT-PCR assay is a sensitive method which circumvents the problems associated with PCR amplification of RNA. Using this assay, we demonstrated that three donors infected by the same index case all have similar patterns of viremia.

Full text

Full text is available as a scanned copy of the original print version. Get a printable copy (PDF file) of the complete article (1.2M), or click on a page image below to browse page by page. Links to PubMed are also available for Selected References.

Images in this article

Click on the image to see a larger version.

Selected References

These references are in PubMed. This may not be the complete list of references from this article.
  • Alter HJ, Purcell RH, Shih JW, Melpolder JC, Houghton M, Choo QL, Kuo G. Detection of antibody to hepatitis C virus in prospectively followed transfusion recipients with acute and chronic non-A, non-B hepatitis. N Engl J Med. 1989 Nov 30;321(22):1494–1500. [PubMed]
  • Buell GN, Wickens MP, Payvar F, Schimke RT. Synthesis of full length cDNAs from four partially purified oviduct mRNAs. J Biol Chem. 1978 Apr 10;253(7):2471–2482. [PubMed]
  • Busch MP, Wilber JC, Johnson P, Tobler L, Evans CS. Impact of specimen handling and storage on detection of hepatitis C virus RNA. Transfusion. 1992 Jun;32(5):420–425. [PubMed]
  • Chamot E, Hirschel B, Wintsch J, Robert CF, Gabriel V, Déglon JJ, Yerly S, Perrin L. Loss of antibodies against hepatitis C virus in HIV-seropositive intravenous drug users. AIDS. 1990 Dec;4(12):1275–1277. [PubMed]
  • Choo QL, Kuo G, Weiner AJ, Overby LR, Bradley DW, 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. [PubMed]
  • Cristiano K, Di Bisceglie AM, Hoofnagle JH, Feinstone SM. 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. [PubMed]
  • Farci P, Alter HJ, Wong D, Miller RH, Shih JW, Jett B, Purcell RH. 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. [PubMed]
  • Garson JA, Tedder RS, Briggs M, Tuke P, Glazebrook JA, Trute A, Parker D, Barbara JA, 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. [PubMed]
  • Garson JA, Tuke PW, Makris M, Briggs M, Machin SJ, Preston FE, Tedder RS. Demonstration of viraemia patterns in haemophiliacs treated with hepatitis-C-virus-contaminated factor VIII concentrates. Lancet. 1990 Oct 27;336(8722):1022–1025. [PubMed]
  • Godec MS, Asher DM, Swoveland PT, Eldadah ZA, Feinstone SM, Goldfarb LG, Gibbs CJ, Jr, Gajdusek DC. Detection of measles virus genomic sequences in SSPE brain tissue by the polymerase chain reaction. J Med Virol. 1990 Apr;30(4):237–244. [PubMed]
  • Han JH, Shyamala V, Richman KH, Brauer MJ, Irvine B, Urdea MS, Tekamp-Olson P, Kuo G, Choo QL, 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. [PMC free article] [PubMed]
  • 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. [PMC free article] [PubMed]
  • Kedzierski W, Porter JC. A novel non-enzymatic procedure for removing DNA template from RNA transcription mixtures. Biotechniques. 1991 Feb;10(2):210–214. [PubMed]
  • Kotewicz ML, Sampson CM, D'Alessio JM, Gerard GF. Isolation of cloned Moloney murine leukemia virus reverse transcriptase lacking ribonuclease H activity. Nucleic Acids Res. 1988 Jan 11;16(1):265–277. [PMC free article] [PubMed]
  • Kwok S, Higuchi R. Avoiding false positives with PCR. Nature. 1989 May 18;339(6221):237–238. [PubMed]
  • Loeffelholz MJ, Lewinski CA, Silver SR, Purohit AP, Herman SA, Buonagurio DA, Dragon EA. Detection of Chlamydia trachomatis in endocervical specimens by polymerase chain reaction. J Clin Microbiol. 1992 Nov;30(11):2847–2851. [PMC free article] [PubMed]
  • Myers TW, Gelfand DH. Reverse transcription and DNA amplification by a Thermus thermophilus DNA polymerase. Biochemistry. 1991 Aug 6;30(31):7661–7666. [PubMed]
  • Okamoto H, Okada S, Sugiyama Y, Tanaka T, Sugai Y, Akahane Y, Machida A, Mishiro S, Yoshizawa H, Miyakawa Y, et al. 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]
  • 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]
  • Reed KC, Mann DA. Rapid transfer of DNA from agarose gels to nylon membranes. Nucleic Acids Res. 1985 Oct 25;13(20):7207–7221. [PMC free article] [PubMed]
  • Weiner AJ, Kuo G, Bradley DW, Bonino F, Saracco G, Lee C, Rosenblatt J, Choo QL, Houghton M. Detection of hepatitis C viral sequences in non-A, non-B hepatitis. Lancet. 1990 Jan 6;335(8680):1–3. [PubMed]

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

Formats:

Related citations in PubMed

See reviews...See all...

Cited by other articles in PMC

See all...

Links

Recent Activity

Your browsing activity is empty.

Activity recording is turned off.

Turn recording back on

See more...