Development and validation of a ddPCR assay to detect and quantify tobacco DNA in smoke and smokeless tobacco and tobacco-free products
Marie-Alice Fraiture,
Andrea Gobbo,
Chloé Guillitte,
Sophia Barhdadi,
Céline Gau,
Patrick Philipp,
Lucas Marmin,
Ugo Marchesi,
Daniela Verginelli,
Nina Papazova,
Céline Vanhee,
Nancy H.C. Roosens
Affiliations
Marie-Alice Fraiture
Sciensano, Transversal Activities in Applied Genomics (TAG), rue Juliette Wytsman 14, 1050, Brussels, Belgium
Andrea Gobbo
Sciensano, Transversal Activities in Applied Genomics (TAG), rue Juliette Wytsman 14, 1050, Brussels, Belgium
Chloé Guillitte
Sciensano, Transversal Activities in Applied Genomics (TAG), rue Juliette Wytsman 14, 1050, Brussels, Belgium
Sophia Barhdadi
Sciensano, Medicines and Health Products, rue Juliette Wytsman 14, 1050, Brussels, Belgium
Céline Gau
Laboratoire SCL de Strasbourg, Chemin du routoir, 67400, Illkirch-Graffenstaden, France
Patrick Philipp
Laboratoire SCL de Strasbourg, Chemin du routoir, 67400, Illkirch-Graffenstaden, France
Lucas Marmin
Laboratoire SCL de Strasbourg, Chemin du routoir, 67400, Illkirch-Graffenstaden, France
Ugo Marchesi
Istituto Zooprofilattico Sperimentale del Lazio e della Toscana ''M.Aleandri'', Unità Operativa Semplice a Valenza Direzionale - Ricerca e Controllo degli Organismi Geneticamente Modificati, via Appia Nuova 1411, 00178, Roma, Italy
Daniela Verginelli
Istituto Zooprofilattico Sperimentale del Lazio e della Toscana ''M.Aleandri'', Unità Operativa Semplice a Valenza Direzionale - Ricerca e Controllo degli Organismi Geneticamente Modificati, via Appia Nuova 1411, 00178, Roma, Italy
Nina Papazova
Sciensano, Transversal Activities in Applied Genomics (TAG), rue Juliette Wytsman 14, 1050, Brussels, Belgium
Céline Vanhee
Sciensano, Medicines and Health Products, rue Juliette Wytsman 14, 1050, Brussels, Belgium
Nancy H.C. Roosens
Sciensano, Transversal Activities in Applied Genomics (TAG), rue Juliette Wytsman 14, 1050, Brussels, Belgium; Corresponding author. Sciensano, Transversal activities in Applied Genomics (TAG), rue Juliette Wytsman 14, 1050 Brussels, Belgium.
The last decade, smoke and smokeless products claiming to be tobacco-free, including herbal cigarettes and herbal shisha, became available on the European market and gained popularity. This study proposes a new digital droplet PCR (ddPCR) method, designed based on a previously developed real-time PCR (qPCR) method being currently used by the U.S. Food and Drug Administration (FDA) to specifically detect the presence of tobacco DNA in targeting a sequence from the Nicotiana tabacum nia-1 gene. To ensure a harmonized and reliable control by enforcement laboratories, both of these qPCR and ddPCR methods were then evaluated and validated for their compliance to an international standard. First, the performance of these PCR-based methods was successfully assessed as specific and sensitive, and in line with minimum performance requirements from international standard. Secondly, the transferability to external laboratory was confirmed for these PCR-based methods. Finally, the applicability of these PCR-based methods was demonstrated using 7 ground tobacco reference materials from the Tobacco Research Center (TRC) Toronto University as well as 6 commercial smokeless and tobacco-free smoke and smokeless products. Based on this study, the previously developed qPCR method was confirmed as complying with international standard, ensuring a efficient and harmonize use by enforcement laboratories for tobacco control on the European market. Moreover, this study proposed to enforcement laboratories the possibility to use a ddPCR method, enabling the simultaneous detection and absolute quantification of tobacco DNA as well as a limited impact of PCR inhibitors.