title: Result variation and efficiency kinetics in real-time PCR.
abstract: Fluorescent monitoring of DNA amplification is the basis of real-time PCR. Absolute quantification can be achieved using a standard curve method. The standard curve is constructed by amplifying known amounts of standards under identical conditions to that of the samples.The objective of the current study is to propose a mathematical model to assess the acceptability of PCR results. Four commercial standards for HCV-RNA (hepatitis C virus RNA) along with 6 patient samples were measured by real-time PCR, using two different RT-PCR reagents. The standard deviation of regression (Sy,x) was calculated for each group of standard and compared by F-Test. The efficiency kinetics was computed by logistic regression, c2 goodness of fit test was preformed to assess the appropriateness of the efficiency curves.Calculated efficiencies were not significantly different from the value predicted by logistic regression model. Reactions with more variation showed less stable efficiency curves, with wider range of amplification efficiencies.Amplification efficiency kinetics can be computed by fitting a logistic regression curve to the gathered fluorescent data of each reaction. This model can be employed to assess the acceptability of PCR results calculated by standard curve method.
authors.affiliation: Department of Pathology, Imam Hospitals Complex, School of Medicine, Tehran University of Medical Sciences, Tehran, Iran.
authors.forename: Reza
authors.initials: R
authors.lastname: Shahsiah
authors.forename: Alireza
authors.initials: A
authors.lastname: Abdollahi
authors.forename: Farid
authors.initials: F
authors.lastname: Azmoudeh Ardalan
authors.forename: Mohammad Taghi
authors.initials: MT
authors.lastname: Haghi-Ashtiani
authors.forename: Issa
authors.initials: I
authors.lastname: Jahanzad
authors.forename: Mohsen
authors.initials: M
authors.lastname: Nassiri Toosi
mesh_terms: D016009:Chi-Square Distribution; D004261:DNA Replication; D004279:DNA, Viral / Q000096:biosynthesis*; D005453:Fluorescence; D016174:Hepacivirus / Q000235:genetics*; D006801:Humans; D007700:Kinetics; D016015:Logistic Models; D016133:Polymerase Chain Reaction / Q000592:standards*; D011786:Quality Control; D012367:RNA, Viral / Q000032:analysis*; D011933:Reagent Kits, Diagnostic / Q000592:standards; D012015:Reference Standards; D015203:Reproducibility of Results
chemical_list: D004279:DNA, Viral; D012367:RNA, Viral; D011933:Reagent Kits, Diagnostic