ITS2 secondary structure data improves authentication of Moringa oleifera tea products when using with DNA barcoding

Adulteration is a severe issue affecting the herbal industry. Therefore, a robust tool is needed to address this problem. In the current study, Moringabased products (tea) authentication was conducted using DNA barcoding. Two different barcode regions, rbcL and ITS2, were investigated in terms of their effectiveness in authenticating the herbal products. To proceed with the DNA barcoding, a good quality of gDNA is a prerequisite for PCR amplification. Hence, two lysis buffers, PL1 and PL2, were compared to obtain good quality gDNA. Results revealed that a higher gDNA yield was obtained from the fresh plants using PL2, but a lower gDNA yield was obtained for the tea products except for sample P3. The PCR reaction was successfully conducted by amplifying two different barcodes, rbcL and ITS2. The amplicon size for the fresh plant was 643 bp for rbcL and 404 bp for ITS2. In contrast, the generated rbcL amplicon sizes for herbal tea products were 672 bp for P1, 679 bp for P2, 679 bp for P3, and 674 bp for P4. For rbcL and ITS2 amplicon sizes were 395 bp for P1, 406 bp for P2, 398 bp for P3, and 413 bp for P4. The amplified PCR products were analyzed using bioinformatic tools. The neighbour-joining (NJ) analysis for the rbcL barcode indicated that the P2, P3, and P4 tea products were categorized in the same clade with the M. oleifera sequence obtained from GenBank. Simultaneously, P1 was clustered individually with other closely related species. The analysis for the ITS2 barcode showed that all samples were grouped in the same clade. Incorporating secondary structure prediction after NJ analysis improved the discrimination between species