Isolation and Barcoding of Trace Pollen-free DNA for Authentication of Honey

Adulteration and mislabeling of honey to mask its true origin have become a global concern. Pollen microscopy, the current gold standard for identifying honey's geographical and plant origins, is laborious, requires extensive training, and fails to identify filtered honey and honey spiked with pollen from a more favorable plant to disguise its origins. We successfully isolated pollen-free DNA from filtered honey using three types of adsorbents: (i) anti-dsDNA antibodies coupled to magnetic microspheres; (ii) anion-exchange adsorbent; and (iii) ceramic hydroxyapatite. The internal transcribed spacer 2 region of the captured pollen-free DNA was polymerase chain reaction-amplified and subjected to next-generation sequencing. Using an in-house bioinformatics pipeline, initial experiments showed that anion exchange had the greatest capacity to capture trace pollen-free DNA, and it was successfully applied to isolate DNA from five honey samples. Enrichment of trace pollen-free DNA from filtered honey samples opens a new approach for identifying the true origins of honey.