Specific detection and quantification of Aspergillus flavus and Aspergillus parasiticus in wheat flour by SYBR® Green quantitative PCR Noelia Sardiñas a , Covadonga Vázquez a , Jéssica Gil-Serna a ,M a Teresa González-Jaén b , Belén Patiño a, ⁎ a Dpto. Microbiología III, Universidad Complutense de Madrid, Madrid, Spain b Dpto. Genética, Universidad Complutense de Madrid, Madrid, Spain abstract article info Article history: Received 23 June 2010 Received in revised form 8 November 2010 Accepted 30 November 2010 Available online xxxx Keywords: Aspergillus parasiticus Aspergillus flavus Aflatoxins Wheat flour qPCR Aflatoxins are important mycotoxins that represent a serious risk for human and animal health. These mycotoxins are mainly produced by Aspergillus flavus and Aspergillus parasiticus, two closely related species with different array of aflatoxins. In this work, two specific quantitative PCR (qPCR) assays were developed to detect and quantify both species in wheat flour using primers based on the multicopy ITS2 rDNA target sequence. The species specificity of the assays was tested in a wide range of strains of these species and others colonizing the same commodities. The sensitivity of the assay was estimated in 2.5 pg/reaction in both species. Discrimination capacity for detection and relative quantification of A. flavus and A. parasiticus DNA were analyzed using samples with DNA mixtures containing also other fungal species at different ratios. Both qPCR assays could detect spore concentrations equal or higher than 10 6 spores/g in flour samples without prior incubation. These assays are valuable tools to improve diagnosis at an early stage and in all critical control points of food chain integrated in HACCP strategies. © 2010 Elsevier B.V. All rights reserved. 1. Introduction Aflatoxins are the most potent natural carcinogens known (JECFA, 1997) and they are recognized as a possible human carcinogen (group 1A) by International Agency of Research of Cancer (IARC, 1993). Additionally, they have hepatotoxic and immunosuppressive proper- ties which cause acute liver damage, liver cirrhosis, tumour induction and teratogenesis (Chu, 1991). These mycotoxins are produced by several Aspergillus species which contaminate food and raw products. Because of the risk these represent for human and animal health, they are under stringent regulation (Commission of the European Communities, 2006), obliging the destruction of contaminated agricultural products which results in significant economic losses. Aspergillus flavus and Aspergillus parasiticus, both belonging to the section Flavi, are the two major aflatoxin-producing species (Bennett and Klich, 2003; Horn, 2007). These species contaminate numerous food commodities including cereals (Pittet, 1998), pistachios, nuts and peanuts (Jelinek et al., 1989), spices (Bartine and Tantaoui-Elaraki, 1997) and figs (Doster et al., 1996; Färber et al., 1997) in warm climates where they may produce aflatoxins at different points of the food chain, such as preharvest, processing, transportation or storage (Ellis et al., 1991). These species have different toxigenic profiles: A. flavus produces aflatoxin B 1 (excreted in breast milk as M 1 )B 2 , cyclopiazonic acid, aflatrem, 3-nitropropionic acid, sterigmatocystin, versicolorin A and aspertoxin, whereas A. parasiticus produces aflatoxin B 1 ,B 2 ,G 1, G 2 and versicolorin A (Wilson et al., 2002). Discrimination of these two species from each other or from 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. These methods have been used for the detection of aflatoxigenic strains of A. flavus and A. parasiticus.(Chen et al., 2002; Criseo et al., 2001; Färber et al., 1997; González-Salgado et al., 2008; Mayer et al., 2003; Sardiñas et al., 2010; Shapira et al., 1996; Somashekar et al., 2004; Sweeney et al., 2000; Zachová et al., 2003). Quantitative PCR (qPCR) has solved the limitations of conventional PCR, providing a tool for accurate and sensitive quantification of target DNA. The chemistries more widely used to develop qPCR assays are DNA- associating dyes (SYBR® Green I) or fluorescently labelled sequence- specific oligoprobes (TaqMan® oligoprobes) (Mackay et al., 2007). The lower cost of qPCR based on SYBR® Green provides an advantage for choice of this method for routine control analyses of commodities. However, SYBR® Green binds nonspecifically to double helix and this may involve a loss of specificity if primers–dimers or nonspecific fragments are present (Kubista et al., 2006). Because of this, we must consider the target sequence used to design the primers because it could condition the power of discrimination and the sensitivity of the assay. Most PCR assays for detection and quantification of mycotoxigenic fungi have been developed using as a target single copy mycotoxin International Journal of Food Microbiology xxx (2010) xxx–xxx ⁎ Corresponding author. Departamento de Microbiología III. Facultad de Biología. Universidad Complutense de Madrid. 28040-Madrid, Spain. Tel.: +34 913 944 969; fax: +34 913 944 964. E-mail address: belenp@bio.ucm.es (B. Patiño). FOOD-05374; No of Pages 5 0168-1605/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.ijfoodmicro.2010.11.041 Contents lists available at ScienceDirect International Journal of Food Microbiology journal homepage: www.elsevier.com/locate/ijfoodmicro Please cite this article as: Sardiñas, N., et al., Specific detection and quantification of Aspergillus flavus and Aspergillus parasiticus in wheat flour by SYBR® Green quantitative PCR, Int. J. Food Microbiol. (2010), doi:10.1016/j.ijfoodmicro.2010.11.041