Development of nicotiana-specific molecular markers and their application in a loop-mediated isothermal amplification assay

Abstract

An effective identification method for detecting illegal goods involving raw tobacco material is crucial for tobacco monopolies to conduct surveillance. We developed Nicotiana-specific molecular markers to determine whether seized goods contain raw tobacco material. The sequence data for genes related to the nicotine metabolism pathway and genomic data from the public Solanaceae database were used to establish Nicotiana-specific molecular markers. These markers were determined by experimentally verifying 17 types of nontobacco plant material and 91 types of tobacco material belonging to 11 sections of 3 subgenera. Two reliable Nicotiana-specific markers, Ntsp027 and Ntsp151, were selected from among the 209 newly developed markers. The results indicated that the primers corresponding to these two markers can amplify the target fragments in the 91 types of Nicotiana material without amplification of any PCR products in the 17 types of non-Nicotiana material. Furthermore, utilizing the marker Ntsp151, we verified the efficacy of the loop-mediated isothermal amplification (LAMP) assay in authenticating tobacco material. The identification of 21 tea-cigarette products via the combination of GC‒MS, a Nicotiana-specific molecular marker and LAMP methods underscores the utility of Nicotiana-specific DNA markers in determining whether illegal goods contain raw tobacco material. Our results indicate an impressive accuracy rate of 100%, which is consistent with the reliability assessment, underscoring the accuracy of these markers in effectively identifying tobacco material. Our findings can significantly augment the capacity for surveillance and anticounterfeiting efforts by aiding the fight against illicit trade and ensuring the integrity of all tobacco-related products in the market.