Quantitation of HIV-1 by real-time PCR with a unique fluorogenic probe.
Quantitation of HIV-1 specific RNA and DNA is pivotal to understanding the pathophysiology of HIV-1 diseases. A method has been developed for quantitation of HIV-1 DNA/RNA by real-time PCR using a unique fluorogenic primer-probe adduct known as scorpion. The probe hybridises to the extension of the adjoining primer intramolecularly, a process kinetically and thermodynamically more favourable than the conventional bimolecular probe-target hybridisation. Data presented in this paper indicate that the scorpion assay is extremely robust and is quite comparable to beacon-based assays. The scorpion assay is also comparable to quantitative competitive PCR (QC--PCR) assays but requires only a fraction of time and effort. Additionally, the dynamic range of the scorpion assay is several log-fold higher than the conventional end point PCR assays. As few as ten copies of vDNA can be detected in the presence of a large excess of exogenously added genomic DNA. Limiting dilution analysis indicates that the assay is capable of detecting a single copy of the viral template. Thus, the scorpion assay presents a specific and sensitive approach for quantitation of DNA/RNA templates by real-time PCR.
Saha
B K
BK
Department of Pathology, University of Alabama at Birmingham, 619 South 19th St. SW-W287, Birmingham, AL 35233-7331, USA.
Tian
B
B
Bucy
R P
RP
D003199:Computer Systems; D017931:DNA Primers; D004279:DNA, Viral / Q000032:analysis; D005456:Fluorescent Dyes; D015497:HIV-1 / Q000235:genetics / Q000302:isolation & purification*; D006801:Humans; D016133:Polymerase Chain Reaction / Q000379:methods; D012367:RNA, Viral / Q000032:analysis; D015203:Reproducibility of Results
D017931:DNA Primers; D004279:DNA, Viral; D005456:Fluorescent Dyes; D012367:RNA, Viral