Development of a PCR Assay for the Detection of Animal Tissues in Ruminant Feeds

Abstract

The European Community ban on use of meat and bone meal in ruminant feed, as a consequence of the spread of bovine spongiform encephalopathy in Europe, has prompted a number of investigations about the possibility of detecting animal tissues in feedstuff. In this paper, a study on vertebrate primers, designed in the 16S rRNA gene of mitochondrial DNA, is described. These primers were able to amplify fragments that contained between 234 and 265 bp. The fragments were specific for bovine, porcine, goat, sheep, horse, rabbit, chicken, trout, and European pilchard and were confirmed by sequence analysis amplicons. The primers were used in a PCR assay applied to five samples of meat and blood meals of different species and subjected to severe rendering treatments (134.4 to 141.9 degrees C and 3.03 to 4.03 bar for 24 min). The presence of vertebrate tissues was detected in all samples. The assay proved to be rapid and sensitive (detection limit 0.0625%). It can be used as a routine method to detect animal-derived ingredients in animal feedstuff.

Effect of heat and pressure processing on DNA fragmentation and implications for the detection of meat using a real-time polymerase chain reaction

“…appropriate analytical tool. However, there are few Consequently, most systems for the detection of reports examining the design and optimization of DNA from highly processed matrices used amplireal-time PCR assays, although many studies cite cons of approximately 300 bp (Bottero et al 2003; various significant parameters that must be consid- Frezza et al 2003;Tartaglia et al 1998) and the ered during the design process for efficient amplifineed for gel visualization meant that the amplicons cation and therefore sensitive assays. were usually over 130 bp.…”

Biomolecular Approaches for the Identification of Tuna Species

“…DNA extraction: DNA was extracted using the Dneasy Tissue Kit (Qiagen) with minor modifications (Bottero et al, 2003).…”

An alternative method to isoenzyme profile for cell line identification and interspecies cross-contaminations: cytochrome b PCR-RLFP analysis

“…Even if standardized systems for isoenzyme characterization are now commercially available, unfortunately, this method still shows several disadvantages: It is not easily reproducible and does not allow for the identification of avian species, and it is expensive and time consuming (in fact, a panel of at least four isoenzymes must be performed to authenticate a single cell line). Other methods can be used to verify the origin of a cell line: immunological and cytogenetic analyses, specific polymerase chain reactions (PCRs; Parodi et al 2002;Bottero et al 2003;Steube et al 2003;Dalmasso et al 2004;Gao et al 2004;Myers et al 2004;Martellini et al 2005;Ha et al 2006;Cooper et al 2007;Ono et al 2007;), amplified fragment length polymorphism (AFLP; Mueller and Wolfenbarger 1999;Milanesi et al 2003), and DNA fingerprinting. This last technique, based on the analysis of polymorphic markers in the genome, is specifically used to find intraspecies cross-contaminants.…”