High resolution melting (HRM) traceability of black pepper adulteration with papaya seeds and chili

Ceylon pepper or black pepper (Piper nigrum) is the second most important spice grown in Sri Lanka. The prodcut in the market is adulterated with cheap biological agents like dried papaya seeds and chili powder. This research was aimed at establishing a robust DNA fingerprinting strategy called DNA barcoding coupled high resolution melting (Bar-HRM) to report the black pepper authentication results. Here, a modified CTAB method was developed to simultaneously extract good quality DNA from pure samples and admixtures of black pepper and its major adulterants. The universal rbcL, psbA-trnH, and novel gene-specific primers were used to amplify DNA for HRM. According to the results, the melting profiles of pure samples of black pepper, papaya, and chili were clearly separated so that they can be differentiated by HRM analysis. HRM data were further analyzed using Principal Component Analysis (PCA) and the results showed that HRM analysis successfully differentiates three species, separating them into three different clusters. When HRM conditions were applied to admixtures of black pepper, papaya, and chili, the HRM curves of the adulterated samples clearly deviated from the curves of the pure samples. This is the first Bar-HRM system that developed to authenticate black pepper adulteration with papaya seeds and chili.