Development of Bioassays Based on Nucleic Acid Amplification and Next-Generation Sequencing



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Adulteration and mis-labeling of honey to mask its true origin have become a global concern. Pollen microscopy, the current gold standard for identifying the geographical origins of honey, is very laborious and requires extensive training. In addition, filtered honey cannot be identified by pollen examination and can be spiked with pollen from a more favorable plant to disguise its origins. We targeted the nuclear ribosomal ITS2 region of plant DNA, which is known to support genus-level discrimination. We purified pollen-free DNA from honey, filtered or centrifuged to remove all pollens, using three different methods: (i) anti-dsDNA antibodies coupled to magnetic particles; (ii) Q Sepharose anion exchanger; and (iii) ceramic hydroxyapatite, Type I. The ITS2 region of the captured pollen-free DNA was PCR-amplified and subjected to next-generation sequencing (NGS). Q Sepharose showed the greatest capacity to capture trace pollen-free DNA and was applied to DNA isolation from two additional honey samples.
Additionally, using pollen DNA barcoding and NGS, we have developed a method to authenticate Manuka honey, a high-value product native to New Zealand. We targeted the nuclear ribosomal ITS2 region of plant DNA of twenty-one different manuka samples. Using our in-house developed bioinformatics pipeline, we have successfully developed an NGS-based quantitative technique for measuring manuka DNA out of total plant DNA. Enrichment of trace pollen-free DNA from filtered honey samples opens a new approach to identify the true origins of filtered honey samples. The methods developed may be useful in other applications of trace DNA analysis. In another study, we developed an immuno-PCR-based diagnostic platform which couples detection antibodies to self-assembled, ultra-detectable DNA-avidin nanoparticles stabilized with poly(ethylene glycol) to link DNA amplification to target protein concentration. Electrostatic neutralization and steric cloaking of the PCR-amplifiable DNA labels by avidin and PEG coating reduces non-specific “stickiness” and enhances assay sensitivity. We further optimized the detectability of the nanoparticles by incorporating four repeats of a unique synthetic DNA PCR target into each nanoparticle. Using human chorionic gonadotropin hormone (hCG) as a model analyte, this platform was able to quantitate the target hCG protein in femtomolar concentrations using only standard laboratory equipment.



Next-generation sequencing, authentication, immuno-PCR


Portions of this document appear in: Chavan, D., Chen, H., Crum, M., Vu, B., Safari, M., Smith, M., Vekilov, P., Conrad, J. C., Kourentzi, K., & Willson, R. C., (2020). Neutral DNA-avidin nanoparticles as ultrasensitive reporters in immuno-PCR. Analyst, 145(14), 4942–4949.