Browsing by Author "Wijesinghe, W.R.P."

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Adulteration detection of Cinnamomum verum with BarHRM technology
(Faculty of Science, University of Kelaniya, Sri Lanka, 2020) Peiris, M.A.L.M.; Silva, F.H.C.; Wijesinghe, W.R.P.
Sri Lanka is the premier exporter of the true cinnamon (C. verum) in the global market. However, Sri Lankan true cinnamon faces a major threat due to severe competition and adulteration from its substitute cassia (e.g. C. aromaticum). It costs one-third of the price of C. verum but it contains coumarin which is a hepatotoxin at substantial amounts (up to 5%) whereas true cinnamon has only trace amounts (about 0.004%). Therefore, it is paramount to detect adulteration of C. verum from its substitute to protect the reputation of true cinnamon. Chemical and morphological methods can detect the adulteration of C. verum but when it comes to admixtures and value-added products, morphological and chemical methods are not accurate. Hence, the objective of the research was to develop a molecular assay to detect adulteration in commercially available cinnamon products. In this study, DNA sequences of C. verum and C. aromaticum were extracted from the National Center for Biotechnology Information (NCBI) using the keyword “Cinnamomum” and selected barcode region “rbcL”. Gene-specific novel markers were manually designed targeting the identified diagnostic SNP sites. Primer properties were analyzed using NetPrimer software and primers with the best qualities were selected. DNA extraction of cinnamon was done using CTAB method with slight modifications. Real-time PCR and melting curve analysis at 65 ⁰C to 95 ⁰C with a ramping rate of 0.05 ⁰C (Qiagen, Germany) was performed. The melting curve analysis and principal component analysis of the data demonstrated a clear distinction between the two species and results confirm that rbcL gene-specific primers can be used to distinguish C. verum from C. aromaticum. Further, this assay has a great potential to quantify adulterants in commercially available cinnamon samples and extremely valuable for an accurate and rapid adulteration detection of cinnamon value-added products in the global and local market.
High-resolution melting traceability of black pepper adulteration with papaya seeds, chili and/or other potential plants
(Faculty of Science, University of Kelaniya, Sri Lanka, 2020) Herath, H.M.P.; Wijesinghe, W.R.P.
Black pepper (Piper nigrum L.) is a valuable medicinal spice and one of the premier exports in Sri Lanka. The black pepper industry in Sri Lanka faces a major threat due to the adulteration of Ceylon pepper with inferior quality substitutes like papaya seeds and chili powder. Therefore, frequent testing of black pepper products is essential to retain the reputation of Ceylon pepper. Application of morphological and chemical methods have limitations in adulteration detection, especially for admixtures in powdery and processed forms. Available molecular techniques also have limitations of being expensive, time consuming and less reliable. The research was aimed at developing an assay based on DNA barcoding coupled High Resolution Melting Analysis (BarHRM) which is sequencing-free, reliable, yet faster and more economical than DNA barcoding to report the spice authentication results. DNA isolation from dried storage tissues is extremely difficult due to the presence of polysaccharides, polyphenols, proteins like compounds and due to the scarcity of DNA. A modified CTAB method was developed along with a phenol extraction to extract and amplify the required DNA regions from the dried processed admixture of black pepper and its major adulterants. Although the DNA quality of the product varies among different samples, the capability of PCR amplification from any material including powdered admixture affirms the validity of the tests being developed in adulterant detection. Two novel gene-specific primer pairs were designed targeting the assay development and both newly developed rbcL markers were successful in PCR amplification. Subsequently, a relatively novel, high throughput technique called Bar-HRM was applied to detect the black pepper adulteration. According to the results 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 examined using Principal Component Analysis (PCA) and the results showed that HRM analysis successfully differentiates three species, separating them into three different clusters. Then the optimized HRM conditions were applied to admixtures and HRM curves of the adulterated samples were clearly deviated from the pure samples. It could be concluded that developed technique is a very first HRM based high-throughput system to authenticate black pepper adulteration with papaya seeds and chili. Although as a proof of concept this technique was developed to detect papaya and chili adulteration, novel system has the potential to detect other black pepper adulterants as well.