MatK, rbcL, trnH-psbA, ITS, trnL-F, 5S-rRNA and 18S-rRNA have been used for a powerful DNA barcode to yield unambiguous identification( Mishra, 2016). As Newmaster et al., (2013) pointed that there’s no standard for herbal products, and unlabeled herbal products could be a risk for those who consume them. Adulteration and substitution of herbal drugs have become problematic due to the ambiguous identification, species extinction and deforestation (Poornima, 2010; Madhavan et al., 2010; Prakash et al., 2013; Rai et al., 2012).
Newmaster et al. (2013) preferred ITS2 region for medicinal plants as it can detect contamination and substitution of processed plant materials due to the shortness of the sequence and high species resolution, as the nuclear genome has the different rate than plastid genes.
According to mishra (2016) several challenges were popping up for DNA barcode when for the herbal industry, such as lack of reference library and unavailability of voucher specimen (Newmaster et al., 2013).As a solution to provide high authenticity of herbal plants there should be taxonomically validated herbarium vouchers and commercially important unidentified herbal plants should be barcoded by comparing a reference database like NCBI. Further to the author, another problem is that plant barcode was done with plastid regions due to the lack of nucleotide sequences for species discrimination. Multi-locus combining was done as a solution for this problem.
Although, when it comes to PCR technique as mentioned in the subsection 2.4 (Sequences for specific PCR primer design) DNA barcode region was used as a sequence for specific primer design to determine internal variations. Therefore, no need any consideration for multiple loci combination while it is more efficient and cost effective in species identification. Practically specific primer designing by using barcode regions can apply for many samples.
This technique can be used to determine the highly processed end product (a powder or a liquid) of raw materials where the technique, DNA barcode limited to raw materials. In another hand specifically, substitutes and adulterants were determined from a mixture of herbal materials. Likewise, all the requirements of plant identification can be done as this technique is one step above than the DNA barcode.
1.1.1. DNA barcode primers
A collection of previously designed primers from several researches was tabulated in Table 3, which can be used to select the appropriate DNA barcode regions to find internal sequence variations for species specific primer design.