A regression-type classification of TaqMan assay

The minute virus of mice (MVM) TaqMan assay is an endpoint assay that utili zes the ABI prism 7200 fluorometer system to measure the fluorescence signa ls (Rn) generated during a reaction called TaqMan assay. In this assay, the virus-free negative samples produce only low-level background signals, i.e ., small Rn values, while the virus DNA-containing positive samples generat e high signals, i.e., large Rn values. Due to the skewed distribution of Rn values and the fact that the mean Rn values change from plate to plate for both negative and positive samples, it appeared to be impossible to set up the cut-off value that would separate all positive from negative reactions without misclassification using traditional statistical approaches. Using the three-sigma principal and the mean Rn values instead of the individual Rn values, we developed a regression-type classification approach to calcul ate the cut-off value that could not only successfully separate the two gro ups of reactions but also significantly reduce the risk of producing false- positive results. It takes into consideration a number of sources of assay variation including intra-assay, interassay, and intercalibration variation ; it also considers different losses for false-positive and false-negative results. This approach was tested and proven to be useful for the assay res ult interpretation in the MVM TaqMan assay validation.