Standard and Light-Cycler PCR methods for animal DNA species detection in animal feedstuffs

Abstract

In this work four species-specific primers and probes were designed and evaluated for the detection and quantification of bovine, ovine, swine and chicken mitochondrial DNA in feeds. PCR primers were optimized using conventional and Real Time PCR, to detect short species-specific sequences amplifiable from heat treated material. Both methods confirmed the high specificity of the primers designed. Real time quantitative PCR assay allowed the detection of as few as 0.01 ng and 0.05 ng of ovine and bovine genomic DNA, respectively. The detection limit for swine and chicken genomic DNA was 0.5 ng. Sensitivity levels observed in DNA extracted from meat samples processed according to EU legislation were different compared to those in genomic DNAs previously described. They resulted in swine 5 fg of MBM DNA, in chicken 25 ng, in ovine and bovine 50 ng. We confirmed the efficiency and specificity of primers in RT-PCR to detect 0.5% of bovine, ovine, swine and chicken MBM in contaminated feedstuffs. © 2007 Elsevier Ltd. All rights reserved.Keywords: BSE prophylaxis; Rendering material; Species-specific primers; Quantitative real time PCR Industrial relevance: The variant Creutzfeldt Jakob disease is a rare and fatal human neurodegenerative condition clearly linked with the bovine spongiform encephalopathies (BSE) of cattle. The ban of using animal derived protein in animal feeds has efficiently controlled the development of the BSE epidemic. The work presented by Frezza and collaborators is an application of the real time polymerase chain reaction (a standard procedure used in molecular biology also known as RT-PCR) to identify specific DNA of four animal species (bovine, ovine, swine and chicken). This method is applied to the analysis of feeds to detect and eventually estimate the amount of animal derived proteins. The difficult aim to detect DNA derived from heat-treated material was successfully reached using as target short mitochondrial DNA sequences. The method presented could have important application not only in the control of feed production but also in many fields of the food industry as quality and process control.

Biotechnological Uses in Assessment of Some Meat Products Adulteration with Equine Meat

“…It is successfully used for identification of various species of meat (Frezza et al 2008 andMane et al, 2009). Also, Chisholm et al, (2005) developed real-time PCR assays specific for horse and donkey, applicable for detection of low levels of horse or donkey meat in commercial products.…”

DNA based approaches for the detection and identification of bovine meat and bone meal in feeds

“…The condition for lifting the ban on the use of MBM is the development and implementation of a test method that allows the identification of protein species. Different methods, mainly PCR, were already developed for identification of different animal species (12,34) or only ruminant species (14,17,19,28,39,43). In these analyses, samples were used with the addition of MBM above 0.1%.…”

“…Several studies have dealt with the application of polymerase chain reaction (PCR) for the detection of bovine tissue in animal feeds (3,27,28,39,41). In many studies (3,5,6,12,14,26,29,30,32), a species-specific PCR has been developed to identify bovine, porcine and avian tissues in meat and bone meal.…”

Application of cholesterol determination method to indirectly detect meat and bone meals in ruminant feeds

“…3 Therefore, monitoring of the quality and origin of feed components becomes an instrumental factor in the control of BSE, 6 which in turn can guarantee a totally reliable product for the consumers d. 4,9 Thus far, several methods have been proposed for the detection of animal by-products in ruminant feed. [10][11][12][13][14][15][16][17][18][19] Some are based on immunoassay techniques for the detection of animal protein that present a low detection limit, besides being easy to apply. 12,13 Nevertheless, in most cases, immunoassays present a cross reaction with certain compounds, yielding incorrect results or overestimating the concentrations at the time of reading.…”

“…12,13 Nevertheless, in most cases, immunoassays present a cross reaction with certain compounds, yielding incorrect results or overestimating the concentrations at the time of reading. Other methods, such as the polymerase chain reaction (PCR) for the species-specific identification of DNA have also been reported previously, [14][15][16][17][18] using the enzyme-linked immunosorbent assay (ELISA) [20][21][22] or ELISA plus gas chromatography, to detect tissues from the central nervous system. 23,24 The non-chromatographic techniques present a low detection limit, but lack specificity.…”