Robust CT-prediction algorithm for RT-PCR

Real-time PCR is being used increasingly as the method of choice for mRNA quantification, allowing rapid analysis of gene expression from low quantities of starting template. 2009 pandemics increased the need for efficient diagnostic tools that can provide rapid results for a number of pathogens without compromising sensitivity and specificity. Introduction of fluorescence-based Real-Time PCR (RT-PCR) is increasingly used to detect multiple pathogens simultaneously and rapidly by gene expression analysis of PCR amplification data. Real-time PCR data are analyzed after setting an arbitrary threshold that must intersect the signal curve in its exponential phase. The point at which the curve crosses the threshold is called Threshold Cycle (CT) This simple and arbitrary however not an elagant definition of CT value sometimes leads to conclusions that are either false positive or negative. Therefore, the purpose of this paper is to present a stable and consistent alternative approach for the definition and determination of CT value that leads to near zero false positives and negatives.