Development of a PCR protocol to detect patulin producing moulds in food products

Abstract

a b s t r a c tPatulin is a secondary toxic metabolite with important health effects. Several mould species of Penicillium and Aspergillus genera associated with patulin production have been detected in food products. Thus, specific and sensitive methods to detect patulin producing moulds are needed. The aim of this work was to develop a polymerase chain reaction (PCR) method to detect patulin producing moulds in food. 34 patulin producing and 30 non-producing strains belonging to the main species usually reported in food products were used. Patulin production was firstly evaluated by mycellar electrokinetic capillary electrophoresis and high-pressure liquid chromatography-mass spectrometry in all tested strains. Biosynthesis was also used to develop PCR primers derived from the genes involved in patulin. By means of a primer pair based on the isoepoxydon dehydrogenase (idh) gene, a 496-bp amplicon was specifically detected in all the mould strains previously confirmed as patulin producing, regardless of their genus and species. With the developed method it was possible to detect down to 0.5 ng of pure DNA from producing strains and from 1.8 Â 10 2 to 2.7 Â 10 3 conidia g À1 in artificially inoculated foods. No relevant PCR inhibition due to food matrices was observed. The PCR protocol developed could be considered as an appropriate tool to detect patulin producing moulds in food products.

A review of the presence of some food contaminants on the territory of the Republic of Serbia

“…The research conducted in Vojvodina in the period from 2004-2006 indicated that the frequency of contamination of maize with DON was in the range of 40-50% while only 3% of the samples exceeded the maximum allowed concentration of 1 μg/g [64]. Similar results were obtained for wheat and sunflower seed, while the frequency of contamination of soybean was lower than 10% [71].…”

A critical review of producers of small lactone mycotoxins: patulin, penicillic acid and moniliformin

“…Given several references on patulin production by A. terreus, it cannot be excluded that this species, or one of the closely related species, can produce it. Obviously for such small molecules, mass spectrometric (MS) verification is of value, but has to be combined with other data, such as an UV spectrum identical to that of patulin and retention time verification, Bergel et al, 1943Bergel et al, , 1944Diaz and Flannigan, 1997;Escoula, 1974;Lopez-Varga et al, 2007;Paterson, 2004 Luque et al, 2011;Moslem et al, 2010;Oh et al, 1998;Okeke et al, 1993;Paterson, 2004 Boysen et al, 1996;Dombrinck-Kurtzman, 2007;Nielsen et al, 2006Penicillium chrysogenum Chunmei et al, 2013Leistner and Pitt, 1977;Oh et al, 1998;Steiman et al, 1989Penicillium citreonigrum Steiman et al, 1989Penicillium citrinum El-Samawaty et al, 2013Frank, 1977;Oh et al, 1998Penicillium claviforme Afiyatullov et al, 2015Bergel et al, 1943;Borkowska Opacka and Escoula, 1977;Chain et al, 1942;Frisvad and Filtenborg, 1983 see P. vulpinum Dombrinck-Kurtzman, 2007;Eckardt, 1979 Penicillium concentricum II Frisvad andFiltenborg, 1983;Houbraken et al, 2016 see P. coprobium Penicillium coprobium Frisvad and Filtenborg, 1989;Dombrink-Kurtzman and Blackburn, 2005; …”

“…Penicillium cyclopium Efimenko and Yakimov, 1960;Frank, 1972;Leistner and Pitt, 1977 Penicillium dipodomyicola Dombrinck-Kurtzman, 2007;Frisvad et al, 1987Houbraken et al, 2016;Luque et al, Burton, 1949;Burton and Pausacker, 1947 see P. expansum Penicillium expansum Luijk, 1938;Andersen et al, 2004;Anslow et al, 1943;Borkowska Opacka and Escoula, 1977;Casquete et al, 2017;Chunmei et al, 2013;Dombrinck-Kurtzman, 2007;Dombrink-Kurtzman and Blackburn, 2005;El-Samawaty et al, 2013;Frisvad and Filtenborg, 1983;Harwig et al, 1973;Larsen et al, 1998;Okeke et al, 1993;Paster et al, 1995;Paterson, 2004 (IDH gene); Paterson et al, 2003;Sommer et al, 1974;Steiman et al, 1989;Welke et al, 2011Penicillium fellutanum Vismer et al, 1996Penicillium funiculosum Steiman et al, 1989Vismer et al, 1996;Yassin et al, 2010 …”

“…A MS/MS analysis will only yield few fragments for patulin, and thus several other verification tests have to be done to ensure that the metabolite detected is indeed patulin. In the paper by Luque et al (2011) patulin was reported for several known producers and in addition species, such as Aspergillus flavus, Penicillium camemberti, and P. verrucosum. In tests of those fungi for patulin this has never been verified in those species Varga et al, 2011b).…”

“…In tests of those fungi for patulin this has never been verified in those species Varga et al, 2011b). Luque et al (2011) used micellar capillary electrophoresis (retention time) and/or HPLC-MS (retention time and MS) in addition to reporting on a PCR amplicon (496 basepairs) for the FC2/IDH gene involved in the patulin biosynthesis, and despite this, they detected patulin in species that do obviously not produce patulin. Other records of patulin producers are correct, but sometimes the fungi are just synonyms of another species.…”