A multiplex-conventional PCR assay for bovine, ovine, caprine and fish species identification in feedstuffs: Highly sensitive and specific Muhammad Safdar a, * , Yasmeen Junejo a, b a Department of Medical Biology and Genetics, University of Gaziantep, Gaziantep, Turkey b Department of Chemistry, Fatih University, 34500, Büyükçekmece, _ Istanbul, Turkey article info Article history: Received 4 June 2014 Received in revised form 23 August 2014 Accepted 26 August 2014 Available online 6 September 2014 Keywords: Multiplex analysis Species-specific PCR Mitochondrial DNA 18S rRNA Authenticity Feedstuffs abstract A multiplex PCR assay was developed for rapid and reliable identification of bovine, ovine, caprine and fish species in feedstuffs simultaneously. The method merges the use of bovine, ovine, caprine and fish primers that amplify fragments (ovine; 119 bp, caprine; 142 bp, fish; 224 bp and bovine; 271 bp) of the mitochondrial t.glu gene forward and cyt b reverse, 12S rRNA, 12S rRNA, and ATPase subunit 8 genes respectively, and a universal 18S rRNA primers that amplifies a 99 bp from eukaryotic DNA. To evaluate the effect of heat treatment, a severe sterilization condition (133  C at 300 kPa for 20 min) was applied. Multiplex analysis of the reference feedstuff samples showed that the detection limit of the assay was 0.01% for each species. Taken together, all data indicated that this multiplex PCR assay was a simple, rapid, sensitive, specific, and cost-effective detection method for bovine, ovine, caprine and fish species in feedstuffs. © 2014 Elsevier Ltd. All rights reserved. 1. Introduction Transmissible Spongiform Encephalopathies (TSEs) are fatal neuro-degenerative diseases i.e. bovine spongiform encephalopa- thy (BSE) in bovine, variant of CreutzfeldteJakob disease in human (Hueston, 2013) and scrapie in sheep and goat (McIntyre, Vilas, & Gubbins, 2011). To minimize the risks of TSEs to humans and ani- mals, the European Food Safety Authority adopted measurement to restrict the processed animal proteins (PAPs) directly or indirectly in ruminant feed (Commission Decision 1994/381/EC; Commission Regulation 1774/2002). Later on the Annexe IV in Regulation 2003/ 1234/EC amended the TSE Regulation, in the sense that all animal proteins from farmed animals are prohibited for the use in feed- stuffs of farmed animals, due to the lack of animal-specific detec- tion methods. However, since June 2013 a Commission allowed the use of processed animal proteins derived from non-ruminant farmed animals in aqua feed. Commission also provided very strict traceability requirements and analytical controls to be applied in order to prevent cross-contamination of fish feed with ruminant proteins susceptible to transmit TSEs (Commission Regulation, 56/2013). Therefore, currently denaturing laboratories are required to have very sensitive analytical tests based on DNA detection to control the correct implementation of the channeling system. Many analytical methods; chemical, electrophoretical, chro- matographic, and immunological have been used for bovine, ovine, caprine and fish species identification in feeds, with each method having its own limitations (Ch afer-Peric as, Maquieira, Puchades, Miralles, & Moreno, 2011; Sentandreu & Sentandreu, 2014). Several researchers have used conventional gel electrophoresis- based PCR-detection for analysis of meat species in feedstuffs (Chen, Liu, & Yao, 2010; Safdar, 2013; Malgorzata, Piotr, & Agata, 2013). In contrast to conventional PCR techniques, real-time PCR- approaches can quantify minute traces of meat species in feedstuffs (Benedetto, Abete, & Squadrone, 2011; Pegels, Gonz alez, García, & Martín, 2014; Safdar, Junejo, Arman, & Abasıyanık, 2014a). How- ever, the high cost of the equipment and reagents is a matter of concern for applying this technique in most laboratories (Safdar & Abasıyanık, 2013a; Zhang, Fowler, Scott, Lawson, & Slater, 2007). Alternatively, multiplex PCR is a rapid, economical and simple approach for commercial screening of feedstuffs (Dalmasso et al., 2004; Ghovvati, Nassiri, Mirhoseini, Heravi Moussavi, & Javadmanesha, 2009; Safdar, Junejo, Arman, & Abasıyanık, 2014b). Therefore it is the urge of time for the requirement of a tech- nique which endorses simple, sensitive, specific and cost-effective methods to use DNA based commercial analysis and surveillance * Corresponding author. Tel.: þ90 5075125460. E-mail addresses: vet_safdar@yahoo.com, drmuhammadsafdar@yahoo.com (M. Safdar). Contents lists available at ScienceDirect Food Control journal homepage: www.elsevier.com/locate/foodcont http://dx.doi.org/10.1016/j.foodcont.2014.08.048 0956-7135/© 2014 Elsevier Ltd. All rights reserved. Food Control 50 (2015) 190e194