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PCR Identification of Aspergillus Niger and Aspergillus Tubingensis Based on Calmodulin Gene Antonia Susca, Gaetano Stea, Giuseppina Mulé, Giancarlo Perrone

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PCR Identification of Aspergillus Niger and Aspergillus Tubingensis Based on Calmodulin Gene Antonia Susca, Gaetano Stea, Giuseppina Mulé, Giancarlo Perrone PCR identification of Aspergillus niger and Aspergillus tubingensis based on calmodulin gene Antonia Susca, Gaetano Stea, Giuseppina Mulé, Giancarlo Perrone To cite this version: Antonia Susca, Gaetano Stea, Giuseppina Mulé, Giancarlo Perrone. PCR identification of Aspergillus niger and Aspergillus tubingensis based on calmodulin gene. Food Additives and Contaminants, 2007, 24 (10), pp.1154-1160. 10.1080/02652030701546206. hal-00577415 HAL Id: hal-00577415 https://hal.archives-ouvertes.fr/hal-00577415 Submitted on 17 Mar 2011 HAL is a multi-disciplinary open access L’archive ouverte pluridisciplinaire HAL, est archive for the deposit and dissemination of sci- destinée au dépôt et à la diffusion de documents entific research documents, whether they are pub- scientifiques de niveau recherche, publiés ou non, lished or not. The documents may come from émanant des établissements d’enseignement et de teaching and research institutions in France or recherche français ou étrangers, des laboratoires abroad, or from public or private research centers. publics ou privés. Food Additives and Contaminants For Peer Review Only PCR identification of Aspergillus niger and Aspergillus tubingensis based on calmodulin gene Journal: Food Additives and Contaminants Manuscript ID: TFAC-2007-170.R1 Manuscript Type: Special Issue Date Submitted by the 21-Jun-2007 Author: Complete List of Authors: Susca, Antonia Stea, Gaetano; National Research Council (CNR), Institute of Sciences of Food Production (ISPA) Mulé, Giuseppina; National Research Council (CNR), Institute of Sciences of Food Production (ISPA) Perrone, Giancarlo; National Research Council (CNR), Institute of Sciences of Food Production (ISPA) Molecular biology - PCR, Mycology, Risk assessment, Screening - Methods/Techniques: microbial screening Additives/Contaminants: Mycotoxins - fungi, Ochratoxin A Food Types: Dried fruit, Wine, Grapes http://mc.manuscriptcentral.com/tfac Email: [email protected] Page 1 of 20 Food Additives and Contaminants 1 2 3 4 5 1 PCR identification of Aspergillus niger and Aspergillus tubingensis 6 7 2 8 based on calmodulin gene. 9 10 3 11 12 4 Antonia Susca, Gaetano Stea, Giuseppina Mulè and Giancarlo Perrone 13 14 15 5 16 For Peer Review Only 17 6 18 19 7 20 Institute of Sciences of Food Production, ISPA-CNR, Via Amendola 122/0, 70126, Bari, 21 22 8 Italy 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 1 http://mc.manuscriptcentral.com/tfac Email: [email protected] Food Additives and Contaminants Page 2 of 20 1 2 3 4 5 1 ABSTRACT 6 7 2 Aspergillus niger and A. tubingensis , species belonging to section Nigri , are commonly 8 9 3 found in plant products and processed food, such as grapes, cereals, coffee and derived 10 11 12 4 products. These two species are very difficult to differentiate by classical morphological 13 14 5 criteria and some isolates are known to produce ochratoxin A. The exact identification 15 16 6 of these twoFor species Peeris very important Review to avoid overestimation Only of toxicological 17 18 19 7 contamination and related risks. A PCR-based identification and detection assay was 20 21 8 developed as a tool to identify A. niger and A. tubingensis, using molecular differences 22 23 9 obtained by sequencing the calmodulin gene. Two pairs of species-specific primers 24 25 26 10 were designed and empirically evaluated for PCR identification of A. niger and A. 27 28 11 tubingensis. Species-specific PCR products generated by each primer set were 505 bp 29 30 31 12 (A. tubingensis ) and 245 bp ( A. niger ) in length, which could be potentially useful for a 32 33 13 multiplex PCR assay. The sensitivity of this assay was about 10 pg DNA in 25 µl PCR 34 35 14 reaction volume, using pure total DNA of the two species. The method described in this 36 37 38 15 study represents a rapid and reliable procedure to assess the presence in food products 39 40 16 of two ochratoxigenic species of section Nigri . 41 42 17 . 43 44 45 18 46 47 19 48 49 20 KEYWORDS: Aspergillus niger , Aspergillus tubingensis , ochratoxin A, species- 50 51 52 21 specific primers¸ PCR, calmodulin gene 53 54 22 55 56 57 58 59 60 2 http://mc.manuscriptcentral.com/tfac Email: [email protected] Page 3 of 20 Food Additives and Contaminants 1 2 3 4 5 1 Introduction 6 7 2 8 9 3 Aspergillus species belonging to section Nigri are distributed worldwide. Many species 10 11 12 4 of them cause food spoilage, and several are used in the fermentation industry (Bennett 13 14 5 and Klich, 1992), or candidate in the biotechnology industries, such as A. niger which 15 16 6 has been grantedFor the GRASPeer (Generally Review Regarded As Safe) Only status by the Food and Drug 17 18 19 7 Administration of the US Government. Recently, the significance of these species, 20 21 8 commonly known as “black aspergilli” has completely changed since they were 22 23 9 identified as the main fungi responsible for the ochratoxin A (OTA) accumulation in 24 25 26 10 grapes and wine (Cabanes et al. 2002; Da Rocha Rosa et al., 2002; Battilani and Pietri, 27 28 11 2002; Magnoli et al., 2003). OTA is a potent nephrotoxin diffusely distributed in food 29 30 31 12 and feed products such as grains, legumes, coffee, dried fruits, beer, wine, and meats. 32 33 13 Since A. tubingensis and A. niger , together with A. carbonarius , are known to produce 34 35 14 OTA, their exact identification within the A. niger aggregate group of species is very 36 37 38 15 important to avoid overestimation of contamination and toxicological risk (Medina et 39 40 16 al. , 2005; Perrone et al. , 2006a). In spite of this, the identification of species within the 41 42 17 A. niger “aggregate”, a group of species within section Nigri , is still controversial. 43 44 45 18 Molecular studies reduced the number of synonym names of described species within 46 47 19 the black aspergilli and supported the division of this “aggregate” in two to four 48 49 20 morphologically indistinguishable species: A. niger , A. tubingensis , A. foetidus and A. 50 51 52 21 brasiliensis (Kuster van Someren et al., 1991; Accensi et al., 1999; Parenicova et al., 53 54 22 2000; Abarca et al., 2004). These four species are very difficult to differentiate by 55 56 57 23 classical morphological criteria, such as conidial shape, colour and size (Samson et al. , 58 59 24 2004). However, effectiveness in the estimation of OTA contamination is dependent on 60 3 http://mc.manuscriptcentral.com/tfac Email: [email protected] Food Additives and Contaminants Page 4 of 20 1 2 3 4 5 1 the association between species and OTA production, hence the use of primers 6 7 2 identified on genes involved in the metabolic pathway would be more helpful. Although 8 9 3 two primers were recently developed to detect OTA producers by Polymerase Chain 10 11 12 4 Reaction (PCR) (Dao et al., 2005), genes involved in steps of OTA biosynthesis have 13 14 5 not yet been identified. A PCR-based identification and detection of species could be 15 16 6 an useful toolFor for identifying Peer potential Review ochratoxigenic Aspergillus Only spp. The calmodulin 17 18 19 7 gene has proved to be highly useful in discrimination of species belonging to section 20 21 8 Nigri , since it contains some species specific diagnostic traits, suitable for diagnostic 22 23 9 purposes. Molecular differences were exploited to set up a qualitative PCR assay for A. 24 25 26 10 carbonarius and A. japonicus /A. aculeatus detection (Perrone et al., 2004). Moreover a 27 28 11 quantitative real-time PCR assay using TaqMan chemistry was recently set up for A. 29 30 31 12 carbonarius DNA quantification on grapes (Mulè et al., 2006). 32 33 13 34 35 14 The objective of this work was to set up a species-specific PCR assay based on 36 37 38 15 differences in sequences found in the calmodulin gene for an accurate identification of 39 40 16 A. niger and A. tubingensis in pure cultures. 41 42 17 43 44 45 18 Material and methods 46 47 19 Fungal strains 48 49 20 One hundred and fifty strains were analyzed in this study: 10 type-strains belonging to 50 51 52 21 Aspergillus sect. Nigri , A. aculeatus ITEM 7046 (GenBank accession no. for 53 54 22 calmodulin gene sequences, AJ964877), A. brasiliensis ITEM 7048 (AM295175), A. 55 56 57 23 carbonarius ITEM 4503 (AJ964873), A. ellipticus ITEM 4505 (AM117809), A. 58 59 24 foetidus ITEM 4507 (AM419749), A. heteromorphus ITEM 7045 (AM421461), A. 60 4 http://mc.manuscriptcentral.com/tfac Email: [email protected] Page 5 of 20 Food Additives and Contaminants 1 2 3 4 5 1 ibericus ITEM 6600 (AJ971806), A. japonicus ITEM 4497 (AJ582717), A. niger 6 7 2 ITEM 4502 (AJ964872), A. tubingensis ITEM 7040 (AJ964876); 136 strains of “black 8 9 3 aspergilli” isolated from grapes grown in different geographical areas: 1 A. aculeatus , 4 10 11 12 4 A. ibericus, 4 A. brasiliensis, 31 A. carbonarius, 23 A. japonicus , 44 A. niger , 29 A. 13 14 5 tubingensis (Perrone et al., 2006b); and 4 strains as outgroup: 2 A. ochraceus ,1 Botrytis 15 16 6 cinerea (ITEMFor 3715) Peerand 1 Saccharomyces Review cerevisae (ITEM Only 6904). All strains were 17 18 19 7 obtained from the culture collection of the Institute of Sciences of Food Production, 20 21 8 with accession number ITEM.
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