Detection of Carica papaya Adulteration in Piper nigrum Using Chloroplast DNA Marker-Based PCR Assays

The spice made from the fruits of Piper nigrum (PN) has economic and medicinal importance. Due to the high demand, as well as export and trade, the quality is threatened by the mixture of cheap and morphologically similar materials, mainly Carica papaya (CP) seeds. The primary objective of this study was to develop a PCR assay to detect CP adulteration and also confirm the presence of PN. For that, PN and CP specific primers were designed from the unique nucleotide sequence regions in the chloroplast genomes of both plants. Sanger sequencing of the specific amplicon revealed that PN primer sequences fall within the region of the rps16 gene and CP primer sequences fall within the region of the trnK-UUU (CP) gene. The designed primers were subjected to optimization of PCR conditions, sensitivity, and cross-reactivity assay. We have sequentially optimised simplex, duplex, and digital PCR (dPCR) to detect the lower quality and quantity of the DNA extracted from blended formulations. The primers for PN and CP were found to be specific and sensitive enough to amplify the target sequence down to 0.1 ng of total genomic DNA. Simplex, duplex, and dPCR assays were able to detect 1.0% (w/w) adulteration of CP seeds in a simulated blended formulation. Thus, the developed primers and assay can be used for the detection of adulteration of CP seeds in PN products.