Development of a Molecular Detection System for Argemone spp.

The analysis of nucleotide sequences of the Argemone genus samples by bioinformatics methods was aimed at the study of phylogenetic relationships of species within the genus, at the development of genus-specific primer pairs, and TaqMan-probe and their verification in silico. Molecular genetic studies by the polymerase chain reaction method in the “real-time” mode (RT-PCR) were devoted to checking the “performance” and specificity of the developed system of primers and TaqMan-probe using samples of eight species of Argemone and 11 species of other plants. Phylogenetic analysis using internal transcribed spacer 1 (ITS1) sequences confirmed previous phylogenetic studies and improved understanding of relationships within the genus. Before the in vitro test of the developed system of primers and TaqMan-probe, the ability of the extracted DNA to be amplified by the RT-PCR method using the system of primers and the TaqMan-probe to the 18S rRNA gene of eukaryotes was checked to exclude pseudo-negative results during the further verification of specificity. The conditions for RT-PCR were selected, namely, temperatures and times of denaturation, hybridization, elongation, and the number of cycles. The developed system of primers and TaqMan-probe for the RT-PCR method demonstrated high species specificity and sensitivity. Amplification was noted only in DNA samples of eight Argemone species, while PCR analysis of other plant species and negative controls showed no amplification. The detection limit of the developed system was determined to be 0.01%. It is proposed that this marker system be used to detect falsification in food and other products, particularly Argemone contamination in mustard or coconut oil.