A duplex PCR assay for authentication of Ocimum basilicum L. and Ocimum tenuiflorum L in Tulsi churna

The primary requirement for any valuable herbal product is an authentic plant species used in its formulation. In this study, we have attempted to develop a simple and cost effective PCR method for the identification of Ocimum basilicum and Ocimum tenuiflorum. For this purpose, we developed species-specific primers and PCR assays. The primers were designed from the chloroplast genome sequences, PCR methods were optimized and further tested for specificity and sensitivity. Methods have been validated using simulated blended materials to mimic the possible admixtures. Primers of both the species are found to be specific and sensitive enough to amplify 0.1 ng of DNA for O. basilicum whereas 0.5 ng of O. tenuiflorum. Duplex PCR assay is also optimized where, one can detect both the species in a single PCR reaction. The sensitivity of primers was further improved up to approximately 10-100 times with digital PCR assay. Chromatographic methods based on conventional chemical makers of Ocimum have also been investigated. Market samples labelled as 'Tulsi powder' were tested using PCR assay and chromatographic methods. Ursolic acid and oleanolic acid was detected in all the market samples whereas eugenol/methyleugenol has been detected inconsistently in the market samples. A comparison of results from molecular and chromatographic authentication methods suggested that both methods should be used in analysis to achieve maximum fidelity for the samples where there is a doubt. However, for the larger sample size, a rapid and cost-effective viable strategy is required. A duplex PCR assay developed in the current study is enough for the authentication of two Ocimum species, O. basilicum and O. tenuiflorum.