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

摘要

A simple and specific real-time PCR assay based on TaqManp technology has been developed for the identification of cultured tobacco (Nicotiana tabacum) in various commodities such as cigars, cigarettes and reconstituted tobacco. The TaqManp 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 TaqManp 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 DNeasyp Plant Mini kit and the Qiagen Gentrap Puregenep 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[trade mark sign] system had to be implemented to obtain amplifiable DNA.