title: Development of a real-time PCR assay for Penicillium expansum quantification and patulin estimation in apples.
abstract: Due to the occurrence and spread of the fungal contaminants in food and the difficulties to remove their resulting mycotoxins, rapid and accurate methods are needed for early detection of these mycotoxigenic fungi. The polymerase chain reaction and the real time PCR have been widely used for this purpose. Apples are suitable substrates for fungal colonization mostly caused by Penicillium expansum, which produces the mycotoxin patulin during fruit infection. This study describes the development of a real-time PCR assay incorporating an internal amplification control (IAC) to specifically detect and quantify P. expansum. A specific primer pair was designed from the patF gene, involved in patulin biosynthesis. The selected primer set showed a high specificity for P. expansum and was successfully employed in a standardized real-time PCR for the direct quantification of this fungus in apples. Using the developed system, twenty eight apples were analyzed for their DNA content. Apples were also analyzed for patulin content by HPLC. Interestingly, a positive correlation (R(2) = 0.701) was found between P. expansum DNA content and patulin concentration. This work offers an alternative to conventional methods of patulin quantification and mycological detection of P. expansum and could be very useful for the screening of patulin in fruits through the application of industrial quality control.
authors.affiliation: Université Saint-Joseph, Centre d'Analyses et de Recherche (Faculté des Sciences), Campus des Sciences et Technologies, Mar Roukos, Mkallès, P.O Box 11-514, Riad El Solh, 1107 2050 Beirut, Lebanon; INRA, UMR 1331 Toxalim, Research Centre in Food Toxicology, 180 Chemin de Tournefeuille, F-31027 Toulouse, Cedex, France; Université de Toulouse III, ENVT, INP, UMR 1331, Toxalim, F-31076, Toulouse, France.
authors.forename: Joanna
authors.initials: J
authors.lastname: Tannous
authors.affiliation: Laboratory of Microorganisms and Food Irradiation, Lebanese Atomic Energy Commission-CNRS, P.O. Box 11-8281, Riad El Solh, 1107 2260 Beirut, Lebanon. Electronic address: a.atoui@cnrs.edu.lb.
authors.forename: Ali
authors.initials: A
authors.lastname: Atoui
authors.affiliation: Université Saint-Joseph, Centre d'Analyses et de Recherche (Faculté des Sciences), Campus des Sciences et Technologies, Mar Roukos, Mkallès, P.O Box 11-514, Riad El Solh, 1107 2050 Beirut, Lebanon.
authors.forename: André
authors.initials: A
authors.lastname: El Khoury
authors.affiliation: Université Saint-Joseph, Centre d'Analyses et de Recherche (Faculté des Sciences), Campus des Sciences et Technologies, Mar Roukos, Mkallès, P.O Box 11-514, Riad El Solh, 1107 2050 Beirut, Lebanon.
authors.forename: Sally
authors.initials: S
authors.lastname: Kantar
authors.affiliation: Université Saint-Joseph, Centre d'Analyses et de Recherche (Faculté des Sciences), Campus des Sciences et Technologies, Mar Roukos, Mkallès, P.O Box 11-514, Riad El Solh, 1107 2050 Beirut, Lebanon.
authors.forename: Nader
authors.initials: N
authors.lastname: Chdid
authors.affiliation: INRA, UMR 1331 Toxalim, Research Centre in Food Toxicology, 180 Chemin de Tournefeuille, F-31027 Toulouse, Cedex, France; Université de Toulouse III, ENVT, INP, UMR 1331, Toxalim, F-31076, Toulouse, France.
authors.forename: Isabelle P
authors.initials: IP
authors.lastname: Oswald
authors.affiliation: INRA, UMR 1331 Toxalim, Research Centre in Food Toxicology, 180 Chemin de Tournefeuille, F-31027 Toulouse, Cedex, France; Université de Toulouse III, ENVT, INP, UMR 1331, Toxalim, F-31076, Toulouse, France.
authors.forename: Olivier
authors.initials: O
authors.lastname: Puel
authors.affiliation: Université Saint-Joseph, Centre d'Analyses et de Recherche (Faculté des Sciences), Campus des Sciences et Technologies, Mar Roukos, Mkallès, P.O Box 11-514, Riad El Solh, 1107 2050 Beirut, Lebanon.
authors.forename: Roger
authors.initials: R
authors.lastname: Lteif
mesh_terms: D002851:Chromatography, High Pressure Liquid; D017931:DNA Primers; D005516:Food Microbiology; D005638:Fruit / Q000737:chemistry / Q000382:microbiology*; D027845:Malus / Q000737:chemistry / Q000382:microbiology*; D010365:Patulin / Q000032:analysis* / Q000235:genetics; D010407:Penicillium / Q000235:genetics / Q000302:isolation & purification*; D011786:Quality Control; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D016415:Sequence Alignment
chemical_list: D017931:DNA Primers; D010365:Patulin
keywords: Apples; IAC; Patulin; Penicillium expansum; Real-time PCR (SybrGreen); Specific primers