The detection of horse and donkey using real-time PCR J. Chisholm, C. Conyers, C. Booth, W. Lawley, H. Hird * Central Science Laboratory, Sand Hutton, York, N. Yorkshire, YO41 1LZ, UK Received 7 November 2004; received in revised form 15 March 2005; accepted 18 March 2005 Abstract We have developed real-time PCR assays specific for horse and donkey, applicable to the detection of low levels of horse or don- key meat in commercial products. Primers, designed to the mitochondrial cytochrome b gene, were 3 0 mismatched to closely related and other commercial species. Amplification of non-target species DNA was prevented by truncation of primers at the 5 0 position, thereby conferring complete specificity. Both assays were highly sensitive and detected the presence of 1 pg of donkey template DNA or 25 pg of horse template DNA when assessed using dilutions of DNA in water. Model food samples, spiked with horse or donkey muscle and commercial products containing horse, were successfully tested for the presence of horse or donkey, demonstrating the applicability of the assays to food products. Ó 2005 Elsevier Ltd. All rights reserved. Keywords: Real-time PCR; Horse; Donkey; Detection 1. Introduction Food labelling regulations require the identity of meat in meat products to be accurately labelled. This has resulted in a need for tests which will reliably iden- tify the species of meat present in a food sample and which also must be sensitive and robust enough to be applied to complex food matrices. A range of analytical approaches have been taken to meet these demands, broadly based on detecting either protein or DNA. Of the protein based methods, immunoassay is the most widely used with several companies supplying kits for a range of species. However, proteins are denatured dur- ing heat and pressure processing, making the detection of species present in a processed sample more difficult. DNA has the advantage of being a relatively stable mol- ecule, and is more able to withstand heat processing. DNA methods have commonly been based around the use of species-specific primers in PCR followed by signal detection using gel electrophoresis (Bottero et al., 2003; Meyer, Hofelein, Luthy, & Candrian, 1995; Rodriguez et al., 2003). More recently published reports have focused on the use of specific primers in real-time PCR using TaqManä technology (Brodmann & Moor, 2003; Dooley, Paine, Garrett, & Brown, 2004; Hird et al., 2004; Laube et al., 2003; Mendoza-Romero et al., 2004). This technique utilises fluorescently labelled probes which allow signal generation to be measured in real-time, thus eliminating the need for electrophoresis, end point determination and consequently the subjective analysis of the results. The development of tests which will reliably identify the species of meat present in food has historically fo- cused on species of high economic importance, including pork, beef and chicken. Many different tests now exist for each of these species (Dooley et al., 2004; Lahiff et al., 2001; Laube et al., 2003), whereas the range of assays for the detection for less commonly used meat 0309-1740/$ - see front matter Ó 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.meatsci.2005.03.009 * Corresponding author. Tel.: +44 1904 462585; fax: +44 1904 462111. E-mail address: h.hird@csl.gov.uk (H. Hird). www.elsevier.com/locate/meatsci Meat Science 70 (2005) 727–732 MEAT SCIENCE