Development of a real-time PCR assay for Penicillium expansum quantification and patulin estimation in apples

Joanna Tannous; Ali Atoui; André El Khoury; Sally Kantar; Nader Chdid; Isabelle P. Oswald; Olivier Puel; Roger Lteif

doi:10.1016/j.fm.2015.03.001

Development of a real-time PCR assay for Penicillium expansum quantification and patulin estimation in apples

Joanna Tannous, Ali Atoui, André El Khoury, Sally Kantar, Nader Chdid, Isabelle P. Oswald, Olivier Puel, Roger Lteif

Research output: Contribution to journal › Article › peer-review

41 Scopus citations

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 (R²=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.

Original language	English
Pages (from-to)	28-37
Number of pages	10
Journal	Food Microbiology
Volume	50
DOIs	https://doi.org/10.1016/j.fm.2015.03.001
State	Published - Sep 1 2015
Externally published	Yes

Funding

The authors express their gratitude to S. Peterson, former curator of the ARS Culture Collection, M. Olsen, from the Research and Development Department of the National and Food administration, Sweden and J. Bauer, from the Technical University Munich, for providing some of the fungal strains examined in this study. We are also greatly indebted to C. Afif from the Department of Chemistry at Saint Joseph's University for his kind cooperation in achieving the HPLC analyses. The research work was financially supported by the National Council for Scientific Research (CNRS), Lebanon grant number: 3443 and the Research Council of Saint-Joseph University (Lebanon) grant number: FS38 .

Keywords

Apples
IAC
Patulin
Penicillium expansum
Real-time PCR (SybrGreen)
Specific primers

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10.1016/j.fm.2015.03.001

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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 (R2=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.",

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AU - Tannous, Joanna

AU - Atoui, Ali

AU - El Khoury, André

AU - Kantar, Sally

AU - Chdid, Nader

AU - Oswald, Isabelle P.

AU - Puel, Olivier

AU - Lteif, Roger

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N2 - 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 (R2=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.

AB - 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 (R2=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.

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Development of a real-time PCR assay for Penicillium expansum quantification and patulin estimation in apples

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Keywords

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