Qualitative and quantitative polymerase chain reaction assays for an alfalfa (Medicago sativa)-specific reference gene to use in monitoring transgenic cultivars

Genetically modified (GM) alfalfa (Medicago sativa) was marketed for the first time in 2005. For countries with established thresholds for GM plants, methods to detect and quantify their adventitious presence are required. We selected acetyl CoA carboxylase as a reference gene for the detection and quantification of GM alfalfa. Two qualitative polymerase chain reaction (PCR) assays (Acc1 and Acc2) were designed to detect alfalfa. Both were specific to alfalfa, amplifying DNA from 12 separate cultivars and showing negative results for PCR of 15 nonalfalfa plants. The limits of detection for Acc1 and Acc2 were 0.2 and 0.01%, respectively. A quantitative real-time PCR assay was also designed, having high linearity (r > 0.99) over alfalfa standard concentrations ranging from 100 to 2.0 x 10(5) pg of alfalfa DNA per PCR. The real-time PCR assay was effective in quantifying alfalfa DNA from forage- and concentrate-based mixed diets containing different amounts of alfalfa meal.