Purpose: The purpose of this study was to develop a method to isolate pollen DNA from honey and to determine the suitability of the DNA for use in a real-time PCR assay. Canola DNA primers and probes were used in a model real-time PCR detection system.
Methods: Samples of canola honey were dissolved in distilled water at a temperature of 30-40 °C. Pollen was centrifuged at 4000 x g for 10 minutes, resuspended in distilled water, aliquots pooled and recentrifuged for 4 minutes. The washed pollen was resuspended in QIAGEN Fast Lysis Buffer, and samples transferred to Lysis Tubes (Cat. No. 69534). Following overnight incubation, 200 µl of the sample in lysis buffer was used for DNA purification with the “Standard Protocol 200 mg” of the DNeasy mericonTM Food Kit (Catalog Number 69514) . A real-time PCR reaction containing the purified canola DNA and canola-specific primers was performed to determine the extraction efficiency of pollen DNA and its suitability for use in the mericon PCR assay portfolio.
Results: DNA purified from canola honey was shown to be detected in the real-time assay in a specific and efficient fashion by comparison to canola DNA standard curves using defined amounts of canola DNA. CT of the standard curve dilutions were separated by 3 units over the range 25-34 CT , and the canola DNA CT was 29 + 0.7.
Significance: The DNA purification method presented can be used to reliably and specifically isolate DNA from honey for use in real-time PCR assays for detection of specific genes, such as those found in GM events, in a challenging food matrix.