• 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. Feb 1994; 32(2): 292–300.
PMCID: PMC263027

Rapid and simple PCR assay for quantitation of human immunodeficiency virus type 1 RNA in plasma: application to acute retroviral infection.

Abstract

A method for quantitating human immunodeficiency virus type 1 plasma viremia may be useful in monitoring disease progression and the responsiveness of patients to a therapeutic regimen or vaccine. A quantitative assay for viral RNA in plasma or sera that differs in several aspects from those reported previously was developed. First, whereas conventional reverse transcriptase-PCR assays involve a two-step process and use two enzymes, the method described uses a single enzyme, rTth DNA polymerase, for both reverse transcription and PCR. The reactions are carried out in a single tube and with a single buffer solution with uninterrupted thermal cycling. Second, uracil-N-glycosylase and dUTP are incorporated into the reaction mixtures to ensure that any carryover of DNA from previous amplifications will not compromise quantitation. Third, a quantitation standard is incorporated into each reaction mixture so that differences in amplification efficiency caused by sample interferents, variability in reaction conditions, or thermal cycling can be normalized. To ensure comparable amplification efficiency, the quantitation standard has the same primer-binding regions as the human immunodeficiency virus type 1 target and generates an amplified product of the same size and base composition. The probe-binding region was replaced with a sequence that can be detected separately. Fourth, a colorimetric detection format was modified to provide at least a four-log-unit dynamic range. The quantitative assay requires only a single amplification of the sample and can be completed in less than 8 h. The procedure was used on archival samples to demonstrate the viremic spike in acute infection and the suppressed levels of circulating virus following seroconversion.

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 (1.8M), or click on a page image below to browse page by page. Links to PubMed are also available for Selected References.

Selected References

These references are in PubMed. This may not be the complete list of references from this article.
  • Anderson RE, Lang W, Shiboski S, Royce R, Jewell N, Winkelstein W., Jr Use of beta 2-microglobulin level and CD4 lymphocyte count to predict development of acquired immunodeficiency syndrome in persons with human immunodeficiency virus infection. Arch Intern Med. 1990 Jan;150(1):73–77. [PubMed]
  • Bagnarelli P, Menzo S, Manzin A, Giacca M, Varaldo PE, Clementi M. Detection of human immunodeficiency virus type 1 genomic RNA in plasma samples by reverse-transcription polymerase chain reaction. J Med Virol. 1991 Jun;34(2):89–95. [PubMed]
  • Becker-André M, Hahlbrock K. Absolute mRNA quantification using the polymerase chain reaction (PCR). A novel approach by a PCR aided transcript titration assay (PATTY). Nucleic Acids Res. 1989 Nov 25;17(22):9437–9446. [PMC free article] [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]
  • Clark SJ, Saag MS, Decker WD, Campbell-Hill S, Roberson JL, Veldkamp PJ, Kappes JC, Hahn BH, Shaw GM. High titers of cytopathic virus in plasma of patients with symptomatic primary HIV-1 infection. N Engl J Med. 1991 Apr 4;324(14):954–960. [PubMed]
  • Clementi M, Menzo S, Bagnarelli P, Manzin A, Valenza A, Varaldo PE. Quantitative PCR and RT-PCR in virology. PCR Methods Appl. 1993 Feb;2(3):191–196. [PubMed]
  • Coombs RW, Collier AC, Allain JP, Nikora B, Leuther M, Gjerset GF, Corey L. Plasma viremia in human immunodeficiency virus infection. N Engl J Med. 1989 Dec 14;321(24):1626–1631. [PubMed]
  • Cuypers HT, Bresters D, Winkel IN, Reesink HW, Weiner AJ, Houghton M, van der Poel CL, Lelie PN. Storage conditions of blood samples and primer selection affect the yield of cDNA polymerase chain reaction products of hepatitis C virus. J Clin Microbiol. 1992 Dec;30(12):3220–3224. [PMC free article] [PubMed]
  • Daar ES, Moudgil T, Meyer RD, Ho DD. Transient high levels of viremia in patients with primary human immunodeficiency virus type 1 infection. N Engl J Med. 1991 Apr 4;324(14):961–964. [PubMed]
  • Fahey JL, Taylor JM, Detels R, Hofmann B, Melmed R, Nishanian P, Giorgi JV. The prognostic value of cellular and serologic markers in infection with human immunodeficiency virus type 1. N Engl J Med. 1990 Jan 18;322(3):166–172. [PubMed]
  • Ferre F. Quantitative or semi-quantitative PCR: reality versus myth. PCR Methods Appl. 1992 Aug;2(1):1–9. [PubMed]
  • Gilliland G, Perrin S, Blanchard K, Bunn HF. 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. [PMC free article] [PubMed]
  • Groenink M, Fouchier RA, Broersen S, Baker CH, Koot M, van't Wout AB, Huisman HG, Miedema F, Tersmette M, Schuitemaker H. Relation of phenotype evolution of HIV-1 to envelope V2 configuration. Science. 1993 Jun 4;260(5113):1513–1516. [PubMed]
  • Ho DD, Moudgil T, Alam M. Quantitation of human immunodeficiency virus type 1 in the blood of infected persons. N Engl J Med. 1989 Dec 14;321(24):1621–1625. [PubMed]
  • Holodniy M, Katzenstein DA, Sengupta S, Wang AM, Casipit C, Schwartz DH, Konrad M, Groves E, Merigan TC. Detection and quantification of human immunodeficiency virus RNA in patient serum by use of the polymerase chain reaction. J Infect Dis. 1991 Apr;163(4):862–866. [PubMed]
  • Holodniy M, Kim S, Katzenstein D, Konrad M, Groves E, Merigan TC. Inhibition of human immunodeficiency virus gene amplification by heparin. J Clin Microbiol. 1991 Apr;29(4):676–679. [PMC free article] [PubMed]
  • Jackson JB, Ndugwa C, Mmiro F, Kataaha P, Guay L, Dragon EA, Goldfarb J, Olness K. Non-isotopic polymerase chain reaction methods for the detection of HIV-1 in Ugandan mothers and infants. AIDS. 1991 Dec;5(12):1463–1467. [PubMed]
  • Kageyama S, Yamada O, Mohammad SS, Hama S, Hattori N, Asanaka M, Nakayama E, Matsumoto T, Higuchi F, Kawatani T, et al. An improved method for the detection of HIV antigen in the blood of carriers. J Virol Methods. 1988 Dec;22(2-3):125–131. [PubMed]
  • Kellam P, Boucher CA, Larder BA. Fifth mutation in human immunodeficiency virus type 1 reverse transcriptase contributes to the development of high-level resistance to zidovudine. Proc Natl Acad Sci U S A. 1992 Mar 1;89(5):1934–1938. [PMC free article] [PubMed]
  • Larder BA, Kellam P, Kemp SD. Zidovudine resistance predicted by direct detection of mutations in DNA from HIV-infected lymphocytes. AIDS. 1991 Feb;5(2):137–144. [PubMed]
  • Layne SP, Merges MJ, Dembo M, Spouge JL, Conley SR, Moore JP, Raina JL, Renz H, Gelderblom HR, Nara PL. Factors underlying spontaneous inactivation and susceptibility to neutralization of human immunodeficiency virus. Virology. 1992 Aug;189(2):695–714. [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]
  • Longo MC, Berninger MS, Hartley JL. Use of uracil DNA glycosylase to control carry-over contamination in polymerase chain reactions. Gene. 1990 Sep 1;93(1):125–128. [PubMed]
  • Lori F, di Marzo Veronese F, de Vico AL, Lusso P, Reitz MS, Jr, Gallo RC. Viral DNA carried by human immunodeficiency virus type 1 virions. J Virol. 1992 Aug;66(8):5067–5074. [PMC free article] [PubMed]
  • Mullis KB, Faloona FA. Specific synthesis of DNA in vitro via a polymerase-catalyzed chain reaction. Methods Enzymol. 1987;155:335–350. [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]
  • Nunberg JH, Schleif WA, Boots EJ, O'Brien JA, Quintero JC, Hoffman JM, Emini EA, Goldman ME. Viral resistance to human immunodeficiency virus type 1-specific pyridinone reverse transcriptase inhibitors. J Virol. 1991 Sep;65(9):4887–4892. [PMC free article] [PubMed]
  • Pan LZ, Werner A, Levy JA. Detection of plasma viremia in human immunodeficiency virus-infected individuals at all clinical stages. J Clin Microbiol. 1993 Feb;31(2):283–288. [PMC free article] [PubMed]
  • Pantaleo G, Graziosi C, Fauci AS. New concepts in the immunopathogenesis of human immunodeficiency virus infection. N Engl J Med. 1993 Feb 4;328(5):327–335. [PubMed]
  • Piatak M, Jr, Saag MS, Yang LC, Clark SJ, Kappes JC, Luk KC, Hahn BH, Shaw GM, Lifson JD. High levels of HIV-1 in plasma during all stages of infection determined by competitive PCR. Science. 1993 Mar 19;259(5102):1749–1754. [PubMed]
  • Saiki RK, Gelfand DH, Stoffel S, Scharf SJ, Higuchi R, Horn GT, Mullis KB, Erlich HA. Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase. Science. 1988 Jan 29;239(4839):487–491. [PubMed]
  • Scadden DT, Wang Z, Groopman JE. Quantitation of plasma human immunodeficiency virus type 1 RNA by competitive polymerase chain reaction. J Infect Dis. 1992 Jun;165(6):1119–1123. [PubMed]
  • Semple M, Loveday C, Weller I, Tedder R. Direct measurement of viraemia in patients infected with HIV-1 and its relationship to disease progression and zidovudine therapy. J Med Virol. 1991 Sep;35(1):38–45. [PubMed]
  • St Clair MH, Martin JL, Tudor-Williams G, Bach MC, Vavro CL, King DM, Kellam P, Kemp SD, Larder BA. Resistance to ddI and sensitivity to AZT induced by a mutation in HIV-1 reverse transcriptase. Science. 1991 Sep 27;253(5027):1557–1559. [PubMed]
  • Thompson J, Gillespie D. Molecular hybridization with RNA probes in concentrated solutions of guanidine thiocyanate. Anal Biochem. 1987 Jun;163(2):281–291. [PubMed]
  • Trono D. Partial reverse transcripts in virions from human immunodeficiency and murine leukemia viruses. J Virol. 1992 Aug;66(8):4893–4900. [PMC free article] [PubMed]
  • Urdea MS, Horn T, Fultz TJ, Anderson M, Running JA, Hamren S, Ahle D, Chang CA. Branched DNA amplification multimers for the sensitive, direct detection of human hepatitis viruses. Nucleic Acids Symp Ser. 1991;(24):197–200. [PubMed]
  • Wang AM, Doyle MV, Mark DF. Quantitation of mRNA by the polymerase chain reaction. Proc Natl Acad Sci U S A. 1989 Dec;86(24):9717–9721. [PMC free article] [PubMed]
  • Young KK, Resnick RM, Myers TW. Detection of hepatitis C virus RNA by a combined reverse transcription-polymerase chain reaction assay. J Clin Microbiol. 1993 Apr;31(4):882–886. [PMC free article] [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

  • MedGen
    MedGen
    Related information in MedGen
  • PubMed
    PubMed
    PubMed citations for these articles

Recent Activity

Your browsing activity is empty.

Activity recording is turned off.

Turn recording back on

See more...