Novel reference gene, PKABA1, used in a duplex real-time polymerase chain reaction for detection and quantitation of wheat- and barley-derived DNA.
We report the development of a duplex real-time Polymerase Chain Reaction (PCR) for the simultaneous detection and quantification of wheat- and barley-derived DNA. We used a single primer pair to amplify the single-copy gene PKABA1 from wheat and barley, using minor-groove-binding probes to distinguish between the two cereals. The assay was fully specific, and different wheat and barley cultivars exhibited similar Ct values, indicating stability across cultivars with respect to allelic and copy number composition. The limits of detection were 5 and 10 PCR-forming units for wheat and barley, respectively, making the duplex assay as sensitive as other singleplex reference gene systems published. We were able to detect both wheat and barley simultaneously in real food samples, and the duplex assay is considered to be suitable as an endogenous reference gene system for the detection and quantification of wheat and barley in genetically modified organisms (GMO) and other food and feed analyses.
Rønning
Sissel B
SB
Section for Feed and Food Microbiology, National Veterinary Institute, Ullevålsveien 68, Post Office Box 8156 Dep., 0033 Oslo, Norway.
Berdal
Knut G
KG
Andersen
Charlotte Bøydler
CB
Holst-Jensen
Arne
A
D018744:DNA, Plant / Q000032:analysis*; D001467:Hordeum / Q000235:genetics*; D016133:Polymerase Chain Reaction; D011494:Protein Kinases / Q000235:genetics*; D011786:Quality Control; D012680:Sensitivity and Specificity; D014908:Triticum / Q000235:genetics*
D018744:DNA, Plant; D011494:Protein Kinases