A new real-time PCR method to overcome significant quantitative inaccuracy due to slight amplification inhibition

Background: Real-time PCR analysis is a sensitive DNA quantification technique that has recently gained considerable attention in biotechnology, microbiology and molecular diagnostics. Although, the cycle-threshold (Ct) method is the present "gold standard", it is far from being a standard assay. Uniform reaction efficiency among samples is the most important assumption of this method. Nevertheless, some authors have reported that it may not be correct and a slight PCR efficiency decrease of about 4% could result in an error of up to 400% using the Ct method. This reaction efficiency decrease may be caused by inhibiting agents used during nucleic acid extraction or copurified from the biological sample. We propose a new method (Cy0) that does not require the assumption of equal reaction efficiency between unknowns and standard curve. Results: The Cy0 method is based on the fit of Richards' equation to real-time PCR data by nonlinear regression in order to obtain the bes...

• Type: Journal
• Year: 2008
• Where: BMCBI
• Authors: Michele Guescini, Davide Sisti, Marco B. L. Rocchi, Laura Stocchi, Vilberto Stocchi
• Keywords: BMCBI 2008, Efficiency Decrease, Reaction Efficiency, Real-time Pcr