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IntroductionReal-time PCR has become the technique of choice for absolute and relative nucleic acid quantification. The gold standard quantification method in real-time PCR (Ct) assumes that the compared samples have similar PCR efficiency. In order to overcome inaccuracy in qPCR due to efficiency decrease, outlier detection methods have been developped. Recently a new method (SOD) showed a high sensitivity and specificity to detect outlier runs. Instead, a new DNA quantification method, called Cy0, show better accuracy, even if in presence of decreasing efficiency due to, for example, inhibitor presence. In this paper we compare DNA quantification obtained using Cy0 and Ct methods utilization, related to SOD value.
SOD modelShape based kinetic outlier detection (SOD) was based on the shapes of the amplification curves. In order to fit fluorescence raw data, non�linear regression fitting of 5-parameter Richards function were performed.The shape parameters used were the plateau value of amplification curve (Fmax), tangent straight line slope in inflection point (m) and y-coordinate of inflection point (Yf). SOD value is based on Mahalanobis distance; it is a useful way of determining the similarity of an unknown multivariate sample set to a known one. Then if ?2 > 7.81, we can reject the null hypothesis and establish that the shape of the amplification curve is different from the shape of the standard curve samples, considering all 3 parameters . Cy0 were calculated using the present www.cy0method.org, specifically developed.
ResultsFor each runs, in presence of biological inhibitors such as tannic acid, IgG or quercitin, Cy0 and Ct values have been calculated. In presence of these inhibitors, SOD efficiently marked some of these amplification profiles as outliers and consequentially Dna quantification obtained using Ct was markedly impaired. Instead, Dna quantifications obtained with Cy0 method, were not significantly different from the expected values.
ConclusionOur results demonstrated, for the first time, that in presence of three different type of inhibitors at different concentrations, Cy0 method achieve quantification with accuracy significantly higher than Ct, even if in presence of outliers, detected by SOD.