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Development of an allele-specific PCR (AS-PCR) method for identifying high-methyl eugenol-containing purple Tulsi (Ocimum tenuiflorum L.) in market samples

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Abstract

Background

Ocimum tenuiflorum L. is a highly traded medicinal with several therapeutic values. Green Tulsi and purple Tulsi are two subtypes in O. tenuiflorum and both have the same medicinal properties. Recent reports have revealed that purple Tulsi contains higher quantities of methyl eugenol (ME), which is moderately toxic and potentially carcinogenic. Therefore, we developed an allele-specific PCR (AS-PCR) method to distinguish the green and purple Tulsi.

Methods and result

Using the green Tulsi as a reference, 12 single nucleotide polymorphisms (SNPs) and 10 insertions/deletions (InDels) were identified in the chloroplast genome of the purple Tulsi. The C > T SNP at the 1,26,029 position in the ycf1 gene was selected for the development of the AS-PCR method. The primers were designed to amplify 521 bp and 291 bp fragments specific to green and purple Tulsi, respectively. This AS-PCR method was validated in 10 accessions from each subtype and subsequently verified using Sanger sequencing. Subsequently, 30 Tulsi powder samples collected from the market were subjected to molecular identification by AS-PCR. The results showed that 80% of the samples were purple Tulsi, and only 3.5% were green Tulsi. About 10% of the samples were a mixture of both green and purple Tulsi. Two samples (6.5%) did not contain O. tenuiflorum and were identified as O. gratissimum.

Conclusion

The market samples of Tulsi were predominantly derived from purple Tulsi. The AS-PCR method will be helpful for quality control and market surveillance of Tulsi herbal powders.

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Data availability

All data generated or analysed during this study are included in this published article (and its supplementary information files).

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Acknowledgements

The authors acknowledge the SRM Institute of Science and Technology.

Funding

This work was supported by the SRM-DBT Partnership Platform for Contemporary Research Services and Skill Development in Advanced Life Sciences Technologies [Grant Number BT/PR12987/INF/22/205/2015].

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Authors and Affiliations

  1. Department of Genetic Engineering, College of Engineering and Technology, Faculty of Engineering and Technology, SRM Institute of Science and Technology, SRM Nagar, Kattankulathur, Kanchipuram, Chennai, TN, 603203, India

    Raju Balaji & Madasamy Parani

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  1. Raju Balaji
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  2. Madasamy Parani
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Contributions

R.B: Data curation, Formal analysis, Methodology, Visualization, Writing - original draft. M.P: Conceptualization, Funding acquisition, Project administration, Resources, Supervision, Visualization, Writing - review & editing. All authors have reviewed the final version of the manuscript and approved it for publication.

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Correspondence to Madasamy Parani.

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No animal testing was performed during this study. Ocimum tenuiflorum L. is not a protected or endangered species. Sampling activities were not performed at locations where specific permission is required.

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The study does not involve any animal participants and human subjects; this is not applicable.

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The authors declare no competing interests.

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Balaji, R., Parani, M. Development of an allele-specific PCR (AS-PCR) method for identifying high-methyl eugenol-containing purple Tulsi (Ocimum tenuiflorum L.) in market samples. Mol Biol Rep 51, 439 (2024). https://doi.org/10.1007/s11033-024-09365-0

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