Abstract
Authentication of herbal products and spices is experiencing a resurgence using DNA-based molecular tools, mainly species-specific assays and DNA barcoding. However, poor DNA quality and quantity are the major demerits of conventional PCR and real-time quantitative PCR (qPCR), as herbal products and spices are highly enriched in secondary metabolites such as polyphenolic compounds. The third-generation digital PCR (dPCR) technology is a highly sensitive, accurate, and reliable method to detect target DNA molecules as it is less affected by PCR inhibiting secondary metabolites due to nanopartitions. Therefore, it can be certainly used for the detection of adulteration in herbal formulations. In dPCR, extracted DNA is subjected to get amplification in nanopartitions using target gene primers, the EvaGreen master mix, or fluorescently labeled targeted gene-specific probes. Here, we describe the detection of Carica papaya (CP) adulteration in Piper nigrum (PN) products using species-specific primers. We observed an increase in fluorescence signal as the concentration of target DNA increased in PN-CP blended formulations (mock controls). Using species-specific primers, we successfully demonstrated the use of dPCR in the authentication of medicinal botanicals.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
Similar content being viewed by others
References
Ichim MC (2019) The DNA-based authentication of commercial herbal products reveals their globally widespread adulteration. Front Pharmacol 10:1–9. https://doi.org/10.3389/fphar.2019.01227
Raclariu AC, Heinrich M, Ichim MC, de Boer H (2018) Benefits and limitations of DNA barcoding and metabarcoding in herbal product authentication. Phytochem Anal 29:123–128. https://doi.org/10.1002/pca.2732
Wu HY, Shaw PC (2022) Strategies for molecular authentication of herbal products: from experimental design to data analysis. Chinese Med (United Kingdom) 17:1–15. https://doi.org/10.1186/s13020-022-00590-y
Quan PL, Sauzade M, Brouzes E (2018) DPCR: a technology review. Sensors (Switzerland) 18. https://doi.org/10.3390/s18041271
Beinhauerova M, Babak V, Bertasi B, Boniotti MB, Kralik P (2020) Utilization of digital PCR in quantity verification of plasmid standards used in quantitative PCR. Front Mol Biosci 7:1–13. https://doi.org/10.3389/fmolb.2020.00155
Huggett JF (2020) The digital MIQE guidelines update: minimum information for publication of quantitative digital PCR experiments for 2020. Clin Chem 66:1012–1029. https://doi.org/10.1093/clinchem/hvaa125
Travadi T, Sharma S, Pandit R, Nakrani M, Joshi C, Joshi M (2022) A duplex PCR assay for authentication of Ocimum basilicum L. and Ocimum tenuiflorum L in Tulsi churna. Food Control 137: https://doi.org/10.1016/j.foodcont.2021.108790
Aboul-Maaty NA-F, Oraby HA-S (2019) Extraction of high-quality genomic DNA from different plant orders applying a modified CTAB-based method. Bull Natl Res Cent 43:1–10. https://doi.org/10.1186/s42269-019-0066-1
Porebski S, Bailey LG, Baum BR (1997) Modification of a CTAB DNA extraction protocol for plants containing high polysaccharide and polyphenol components. Plant Mol Biol Report 15:8–15. https://doi.org/10.1007/BF02772108
Clarke JD (2009) Cetyltrimethyl ammonium bromide (CTAB) DNA miniprep for plant DNA isolation. Cold Spring Harb Protoc 2009:pdb-prot5177
Krizman M, Jakse J, Baricevic D, Javornik B, Prosekm M (2006) Robust CTAB-activated charcoal protocol for plant DNA extraction. Acta Agric Slov 87:427–433
Tan LL, Loganathan N, Agarwalla S, Yang C, Yuan W, Zeng J, Wu R, Wang W, Duraiswamy S (2022) Current commercial dPCR platforms: technology and market review. Crit Rev Biotechnol 0:1–32. https://doi.org/10.1080/07388551.2022.2037503
Travadi T, Shah AP, Pandit R, Sharma S, Joshi C, Joshi M (2022) Detection of Carica papaya adulteration in Piper nigrum using chloroplast DNA marker-based PCR assays. Food Anal Methods. https://doi.org/10.1007/s12161-022-02395-z
Sakamoto W, Takami T (2018) Chloroplast DNA dynamics: copy number, quality control and degradation. Plant Cell Physiol 59:1120–1127. https://doi.org/10.1093/pcp/pcy084
Yu N, Xing R, Wang P, Deng T, Zhang J, Zhao G, Chen Y (2022) A novel duplex droplet digital PCR assay for simultaneous authentication and quantification of Panax notoginseng and its adulterants. Food Control 132:108493
Yu N, Han J, Deng T, Chen L, Zhang J, Xing R, Wang P, Zhao G, Chen Y (2021) A novel analytical droplet digital PCR method for identification and quantification of raw health food material powder from Panax notoginseng. Food Anal Methods 14:552–560. https://doi.org/10.1007/s12161-020-01887-0
Xu W, Zhu P, Xin T, Lou Q, Li R, Fu W, Ma T, Song J (2022) Droplet digital PCR for the identification of plant-derived adulterants in highly processed products. Phytomedicine 105:154376. https://doi.org/10.1016/j.phymed.2022.154376
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2023 The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature
About this protocol
Cite this protocol
Shah, A.P., Travadi, T., Sharma, S., Pandit, R., Joshi, C., Joshi, M. (2023). Digital PCR: A Tool to Authenticate Herbal Products and Spices. In: Domingues, L. (eds) PCR. Methods in Molecular Biology, vol 2967. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-3358-8_2
Download citation
DOI: https://doi.org/10.1007/978-1-0716-3358-8_2
Published:
Publisher Name: Humana, New York, NY
Print ISBN: 978-1-0716-3357-1
Online ISBN: 978-1-0716-3358-8
eBook Packages: Springer Protocols