Detection of Nicotiana DNA in Tobacco Products Using a Novel Multiplex Real-Time PCR Assay

• Published in: Journal of AOAC International 2016-07, Vol.99 (4), p.1038-1042
• Main Authors: Korchinski, Katie L, Land, Adrian D, Craft, David L, Brzezinski, Jennifer L
• Format: Article
• Language: English
• Subjects: DNA, Plant - analysis; DNA, Plant - genetics; Multiplex Polymerase Chain Reaction; Tobacco Products - analysis
• ISSN: 1060-3271; 1944-7922
• DOI: 10.5740/jaoacint.16-0008

Establishing that a product contains tobacco is a requirement for the U.S. Food and Drug Administration's regulation and/or prosecution of tobacco products. Therefore, a multiplex real-time PCR method was designed to determine if Nicotiana (tobacco) DNA is present in tobacco products. The PCR method simultaneously amplifies a 73 bp fragment of the cytochrome P450 monoxygenase CYP82E4 gene and 66 bp fragment in the nia-1 gene for nitrate reductase, which are detected using dual-labeled TaqMan probes. The assay is capable of detecting approximately 7.8 pg purified tobacco DNA, with a similar sensitivity for either gene target while incorporating an internal positive control (IPC). DNA was extracted from prepared tobacco products-including chewing tobacco, pipe tobacco, and snuff-or from the cut fill (no wrapper) of cigarettes and cigars. Of the 13 products analyzed, 12 were positive for both tobacco-specific markers and the IPC. DNA was also extracted from the fill of five varieties of herbal cigarettes, which were negative for both tobacco-specific gene targets and positive for the IPC. Our method expands on current assays by introducing a multiplex reaction, targeting two sequences in two different genes of interest, incorporating an IPC into the reaction, and lowering the LOD and LOQ while increasing the efficiency of the PCR.