Real-time TaqMan PCR as a specific and more sensitive alternative to the branched-chain DNA assay for quantitation of simian immunodeficiency virus RNA

C. M. Leutenegger, J. Higgins, T. B. Matthews, Alice F Tarantal, Paul A Luciw, Niels C Pedersen, T. W. North

Research output: Contribution to journalArticle

102 Citations (Scopus)

Abstract

We developed a rapid and highly reproducible assay based on real-time PCR (TaqMan, Applied Biosystems, Foster City, CA) to quantitate simian immunodeficiency virus (SIV) RNA in plasma samples. This assay was compared with the current branched-chain DNA assay (Bayer, Emeryville, CA). Results obtained with the real-time TaqMan PCR assay were comparable to those obtained with the branched-chain DNA assay in overlapping ranges of sensitivities (r = 0.9429, p < 0.05). However, the real-time TaqMan PCR assay was capable of detecting as few as 50 copies of RNA/ml, whereas branched-chain DNA was only sensitive to 1,500 copies of RNA/ml. Therefore, several animals that tested negative by branched-chain DNA were positive by real-time TaqMan PCR. Two false positive tests were also recorded for the branched-chain DNA test. False negative and positive tests were confirmed by cell culture isolation and conventional nested RT-PCR. The SIV TaqMan assay detected a wide range of wild-type, cloned, and recombinant SIV strains with similar amplification efficiency, including SIVmac251, SIVmac239, SIVmac239 containing the 184V mutation in RT, SIV1A11, SIVmac239 delta3, SIVmac-M4, and chimeras (SHIVs) containing specific HIV-1 genes, such as reverse transcriptase (RT-SHIV) or Env (SHIV-E). In conclusion, the high sensitivity, increased specificity, wide dynamic range, simplicity, and reproducibility of the real-time SIV RNA quantitation allow the screening of large numbers of samples and make this method especially suitable for measuring both viral DNA and RNA levels during vaccine and therapy studies.

Original languageEnglish (US)
Pages (from-to)243-251
Number of pages9
JournalAIDS Research and Human Retroviruses
Volume17
Issue number3
DOIs
StatePublished - Feb 10 2001

Fingerprint

Branched DNA Signal Amplification Assay
Simian Immunodeficiency Virus
Real-Time Polymerase Chain Reaction
RNA
DNA
Active Immunotherapy
Cell Separation
RNA-Directed DNA Polymerase
Viral DNA
Viral RNA
HIV-1
Cell Culture Techniques
Sensitivity and Specificity
Polymerase Chain Reaction
Mutation
Genes

ASJC Scopus subject areas

  • Immunology
  • Virology

Cite this

Real-time TaqMan PCR as a specific and more sensitive alternative to the branched-chain DNA assay for quantitation of simian immunodeficiency virus RNA. / Leutenegger, C. M.; Higgins, J.; Matthews, T. B.; Tarantal, Alice F; Luciw, Paul A; Pedersen, Niels C; North, T. W.

In: AIDS Research and Human Retroviruses, Vol. 17, No. 3, 10.02.2001, p. 243-251.

Research output: Contribution to journalArticle

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