Analysis and interpretation of data from real-time PCR trace detection methods using quantitation of GM soya as a model system

Abstract

Recent years have seen an increased interest in DNA trace detection methods involved in many areas of bioanalytical research, such as quantitation of genetically modified (GM) ingredients in food products. There is little in the way of standardisation of data handling from these methods, and the data generated needs to be analysed appropriately if the results are to be interpreted correctly. This paper describes particular aspects of real-time PCR trace detection methods in order to increase the understanding of data generated using this bioanalytical technique. Using the specific example of GM soya detection and quantitation, it focuses on the production of calibration curves based on the mean and individual data values, the interpretation of correlation coefficients, regression techniques, and discusses suitable data analysis arising from simple and more complex experimental designs following transformation. By using the approaches outlined in this paper, more accurate analysis of data from real-time PCR and GM trace detection methods could be achieved.

Sensitive Detection of Multiple Rotavirus Genotypes with a Single Reverse Transcription-Real-Time Quantitative PCR Assay

“…The potential of the approach for the detection of the broadest range of rotavirus strains is further enhanced by the presence in the PCR of a mixture of five forward and two reverse primers in combination with a degenerate TaqMan MGB probe. The method successfully detected the presence of rotavirus in 139 isolates (Table 4), involving 17 different G-P types divided into 7 of human origin (G1P [8], G2P [4], G3P [8], G4P [8], G8P [8], G9P [8], and G12P [8]), 3 of bovine origin (G6P [1], G6P [1] G6P [5], and G6P [11]), and 7 of porcine origin (G2P [13], G3P [6], G4P [22], G5P [7], G5P [13], G9P [13], and G11P [13]). The flexibility of the developed approach did not compromise its sensitivity.…”

“…The viral-RNA concentration associated with each cDNA dilution was recalculated based on the obtained regression equations and was used for calculating the coefficient of variance within each triplicate. cDNA dilutions whose triplicate C T values gave a coefficient of variance lower than 30% were assumed to be within the range of quantification (4,5). In regard to the range of detection, cDNA dilutions that gave a positive C T value in at least two of the triplicates were considered to be within the range of detection.…”

The MIQE Guidelines: Minimum Information for Publication of Quantitative Real-Time PCR Experiments

“…Rather, these guidelines target the reliability of results to help ensure the integrity of the scientific literature, promote consistency between laboratories, and increase experimental transparency. They should be read in conjunction with recent publications that deal in depth with the issue of qPCR standardization (23)(24)(25)(26).…”

Detection and quantification of Tomato mosaic virus in irrigation waters

“…The limit of detection (LOD) for real-time PCR was therefore 4.2 · 10 -10 mg ml -1 . According to Burns Valdivia, and Harris (2004), the concentration value that presents the LOD is where the coefficient of variation (CV) value drops below 50%, and the LOQ is set at a threshold of a CV below 30%. Thus the LOQ for the ToMV-specific real-time PCR was set to 4.2 · 10 -8 mg ml -1 .…”