ITS-based detection and quantification of Aspergillus ochraceus and Aspergillus westerdijkiae in grapes and green coffee beans by real-time quantitative PCR Jéssica Gil-Serna a , Amaia González-Salgado b ,M a Teresa González-Jaén b , Covadonga Vázquez a , Belén Patiño a, ⁎ a Department of Microbiology III, Faculty of Biology, University Complutense of Madrid, José Antonio Novais 2, E 28040 Madrid, Spain b Department of Genetics, Faculty of Biology, University Complutense of Madrid, José Antonio Novais 2, E 28040 Madrid, Spain abstract article info Article history: Received 9 June 2008 Received in revised form 12 January 2009 Accepted 9 February 2009 Keywords: Aspergillus ochraceus Aspergillus westerdijkiae Real-time qPCR OTA Grapes Green coffee beans ITS Aspergillus ochraceus and A. westerdijkiae are considered the most important Ochratoxin A (OTA) producing species included in Aspergillus section Circumdati which contaminate foodstuffs and beverages for human consumption. In this work a real-time quantitative PCR protocol was developed to detect both species using SYBR® Green and primers designed on the basis of the multicopy ITS1 region of the rDNA. The assay had high efficiency (94%) and showed no inhibition by host or fungal DNA other than the target species. The lower detection limit of the target DNA was 2.5 pg/reaction. Accuracy of detection and quantification by qPCR were tested with genomic DNA obtained from green coffee beans and grapes artificially contaminated with spore suspensions of known concentrations. Spore concentrations equal or higher than 10 6 spore/ml could be detected by the assay directly without prior incubation of the samples and a positive relationship was observed between incubation time and qPCR values. The assay developed would allow rapid, specific, accurate and sensitive detection and quantification of A. ochraceus and A. westerdijkiae to be directly used in a critical point of the food chain, before harvesting green coffee and grape berries, to predict and control fungal growth and OTA production. © 2009 Elsevier B.V. All rights reserved. 1. Introduction Ochratoxin A (OTA) is a widespread mycotoxin with nephrotoxic, inmunotoxic, genotoxic and teratogenic properties towards several animal species (Pfohl-Leszkowicz and Manderville, 2007) and has been classified by the International Agency for Research on Cancer as a possible human carcinogen (group 2B) (IARC, 1993). The maximum OTA limits allowed in several food and raw agro-products for human consumption are under legal regulation in the European Union (Commission Regulation, 2006). This mycotoxin occurs in various foodstuffs including cereals and derivatives (Rizzo et al., 2002), coffee (Taniwaki et al., 2003), grapes and grape-products (Varga and Kozakiewicz, 2006), dried fruits (Zinedine et al., 2007) and spices (Rizzo et al., 2002). OTA is a secondary metabolite produced by several fungal species belonging to the Aspergillus and Penicillium genera. Aspergillus ochraceus was the first OTA-producing species described (Van der Merwe et al., 1965) and it is considered an important species con- tributing to OTA contamination of coffee, grapes and cereals (Taniwaki et al., 2003; Magnoli et al., 2007). Other important OTA-producing species of the Section Circumdati are the more recently described A. westerdijkiae and A. steynii (Frisvad et al., 2004; Samson et al., 2006). Discrimination among these species and from other closely related species is difficult when conventional methods based mainly on morphological features are used and requires considerable expertise. The application of DNA-based techniques permits rapid, sensitive and specific detection, necessary to devise strategies to control or reduce fungal mass and toxin production at early and critical stages of the food chain, and they are replacing traditional methods in many areas related with food analyses (Niessen et al., 2005). Real-time quanti- tative PCR (qPCR) has solved the limitations of conventional PCR, providing a tool to accurate and sensitive quantification of target DNA. The most common chemistries, DNA-associating dyes (SYBR Green I) or fluorescently labelled sequence-specific oligoprobes (TaqMan® oligoprobes) are being widely used to develop qPCR assays (Mackay et al., 2007). The lower cost of qPCR based on SYBR Green is an advantage of this method for detection and quantification protocols used in routine analyses of commodities, but it may involve a loss of specificity if primers-dimers or nonespecific fragments are present (Kubista et al., 2006). Because of this, additional controls should be done such as analyzing the reaction products with a melting curve (Ririe et al., 1997). The target sequence used to design the primers is also relevant, because it will condition the power of discrimination and the sensitivity of the assay. Several qPCR assays to detect and quantify ochratoxigenic fungi have been developed using as target International Journal of Food Microbiology 131 (2009) 162–167 ⁎ Corresponding author. E-mail address: belenp@bio.ucm.es (B. Patiño). 0168-1605/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.ijfoodmicro.2009.02.008 Contents lists available at ScienceDirect International Journal of Food Microbiology journal homepage: www.elsevier.com/locate/ijfoodmicro