A real-time polymerase chain reaction (PCR) method for the identification of Nicotiana tabacum in tobacco products

A simple and specific real-time PCR assay based on TaqMan® technology has been developed for the identification of cultured tobacco (Nicotiana tabacum) in various commodities such as cigars, cigarettes and reconstituted tobacco. The TaqMan® assay targets a sequence of the putrescine N-methyltransferase gene family encoding an enzyme that plays a crucial role in the biosynthesis of nicotine. To reduce the possibility of false negatives, universal plant chloroplast primers were also used in a separate real-time PCR reaction to give indication if DNA is amplifiable in the matrix. The TaqMan® assay successfully identified tobacco in over 40 commercial tobacco products, while negative results were obtained from the assay for DNA extracted from a variety of other botanical products. In our study, two commercial DNA isolation kits were used, namely, the Qiagen DNeasy® Plant Mini kit and the Qiagen Gentra® Puregene® kit. They produced good quality DNAs in sufficient quantities for real-time PCR analysis. In a few cases, an additional purification step with the Promega DNA IQ™ system had to be implemented to obtain amplifiable DNA.