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

We developed a rapid and highly reproducible assay based on real-time PCR ( TaqMan, Applied Biosystems, Foster City, CA) to quantitate simian immunodef iciency 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 de tecting 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. Fa lse negative and positive tests were confirmed by cell culture isolation an d 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 m utation in RT, SIV1A11, SIVmac239 delta3, SIVmac-M4, and chimeras (SHIVs) c ontaining specific HIV-1 genes, such as reverse transcriptase (RT-SHIV) or Env (SHIV-E). In conclusion, the high sensitivity, increased specificity, w ide 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 dur ing vaccine and therapy studies.