Quantitative Detection of Species-Specific DNA in Feedstuffs and Fish Meals

Abstract

A sensitive and rapid method for the quantitative detection of bovine-, ovine-, swine-, and chicken-specific mitochondrial DNA sequences based on real-time PCR has been developed. The specificity of the primers and probes for real-time PCR has been tested using DNA samples of other vertebrate species that may also be present in rendered products. The quantitative detection was performed with dual-labeled probes (TaqMan) using absolute quantification with external standards of single species meat-and-bone meals. This method facilitates the detection of 0.01% of the target species-derived material in concentrate feed mixtures and fish meals.

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

“…The RT-PCR is still the only species-specific technique available to quantify and detect sources of animal protein, (Bellagamba et al, 2006;Fumière, Dubois, Baeten, von Host, & Berben, 2006;Krcmar & Rencova, 2005). Used in combination with complementary techniques such as inmunoassay and spectroscopy methods, it remains an important part of the approach used to determine the origin of animal protein present in feeds (Fumière et al, 2006).…”

“…PCR methods in fact reach precision and sensitivity levels difficult to achieve using microscopic examination, or impossible applying immuno-enzymatic assays, because of protein instability in heattreated material (Ansfield, Reaney, & Jackman, 2000;Baeten et al, 2005;Kim et al, 2005). Attempts to establish multiplex PCR (Dalmasso et al, 2004) and real time PCR (RT-PCR) protocols in contaminated food and feedstuff have recently been reported (Krcmar and Rencova, 2005;Lopez-Andreo, Lugo, GarridoPertiera, Prieto, & Puyet, 2005;Mendoza-Romero et al, 2004;Taurai, Schumacher, & Roger, 2005). Compared to conventional PCR, RT-PCR has the advantage of quantifying the small size products (from 66 to 145 bp), amplified from highly degraded source material, like rendered MBM.…”

Authentication of meats from quail (Coturnix coturnix), pheasant (Phasianus colchicus), partridge (Alectoris spp.), and guinea fowl (Numida meleagris) using polymerase chain reaction targeting specific sequences from the mitochondrial 12S rRNA gene

“…The high temperature and pressure treatments used in some meat products may cause fragmentation of DNA, leading to difficulties to obtain reliable results in PCR amplification (Colgan et al, 2001). Because of this, species identification in samples where DNA can be severely degraded must rely on amplification of short DNA targets (Krcmar & Rencova, 2005).…”

A PCR assay for sex determination of yak (Bos grunniens) meat by amplification of the male-specific SRY gene

“…For this reason, the short DNA targets would improve the amplification chances for the DNA extracted from heat-treated or processed meat and meat products in which DNA might be degraded severely (Arslan et al, 2006;Krcmar & Rencova, 2005). In this work, the length of target DNA was shorter than 300-bp (290-and 121-bp), and both pasteurized (72°C, 30 min) and sterilized (121°C, 20 min) meat samples were also tested with the protocol described.…”