A Competitive Polymerase Chain Reaction–Based Approach for the Identification and Semiquantification of Mitochondrial DNA in Differently Heat-Treated Bovine Meat and Bone Meal

Abstract

The risk of bovine spongiform encephalopathy propagation was drastically reduced after the European Union (EU) Health Authorities adopted restrictions involving a ban on animal-derived proteins in the diet of farm animals. Currently, the EU's of cially recommended method for controlling meat and bone meal (MBM) in animal feed is the microscopic method, which involves the identi cation of bone fragments on the basis of their morphological characteristics. Recently, we demonstrated that a polymerase chain reaction (PCR)-based assay can be used for the detection of taxon-speci c DNA in MBM and animal feeds. To ensure the safe rendering of animal by-products, the EU Council requires that this material be treated at 1338C at 300 kPa for 20 min. Here we investigate the relationship between DNA degradation, PCR ampli cation, and MBM heat treatment. With a competitive PCR-based approach, we compare the ampli cation ef ciency of bovine mitochondrial DNA target sequences of different lengths in several heat-treated MBM samples. For our method, a synthetic competitive DNA is used as an internal control for both DNA extraction and PCR reaction. A correlation between an increase in treatment temperature and a reduction in the size of the target sequences suitable for ampli cation was observed, suggesting progressive DNA fragmentation due to the temperature. We show that short amplicons (147 bp) can be used to detect the presence of bovine mtDNA in MBM samples treated according to the current European regulations. The use of such a competitive approach to compare ampli cation ef ciency levels of targets of different lengths might represent a useful tool for the determination of both the amount of MBM in animal feeds and its proper heat treatment.There is evidence that bovine spongiform encephalopathy (BSE) was spread around the world through animal feed containing infected meat and bone meal (MBM) (3, 4). The effective preventive measures taken by the United Kingdom with the introduction of the so-called ''real feed ban'' in 1996 (a ban on animal proteins in farm animal feeds) have led to a rapid reduction in the appearance of new cases in that country (5). Based on such epidemiological evidence, national and international authorities (Of ce International des Epizooties, the World Health Organization, Codex Alimentarius, and the European Commission) have progressively implemented speci c legislation and guidelines regarding the appropriate heat treatment of MBMs (10), the ban of MBMs from ruminant feeds (6), and the removal of speci ed risk materials from slaughtered ruminants (8). Currently, there is a temporary ban on animal meal in feed for all animals intended for human consumption within the European Union (EU), allowing mem- ber states to improve the practical implementation of the European legislation to prevent the spread of BSE (9). Nevertheless, there is currently no reliable, sensitive, and speci c method to detect the presence of the BSE causative agent in feeds. Therefore, only indirect approaches testing...

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

“…Species-specific PCR based assays have previously been reported for the identification of animal derived material in feedstuffs by amplifying mtDNA genes as cytochrome b, ATPase 8, ATPase 6 and 12 S rRNA (Chen, Wen, Ding, Kao, & Kuo, 2003;Herman, 2001;Myers et al, 2001;Rodríguez et al, 2004). Nevertheless, primers here reported were designed to obtain amplicons smaller than 120 bp, detectable in highly degraded material (Frezza et al, 2003;Chiappini et al, 2005). Indeed, during feedstuffs preparation, the DNA is degraded due to the hyperbaric treatment applied (133°C/3 bar for 20 min).…”

“…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. Recently (Frezza et al, 2003), we demonstrated that short amplicons can be used to detect the presence of bovine mitochondrial DNA (mtDNA) in MBM samples treated according to the current European legislation (133°C, 20′, 3 bars) (Dept. of Agr., Food and Rur.…”

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.…”

Development of PCR Primers for the Detection of Porcine DNA in Feed Using mtATP6 as the Target Sequence

“…Because of its compact circular shape, mtDNA is relatively stable and thus highly suitable for DNA detection in heat-treated materials. Nevertheless, mtDNA might be damaged/fragmented during the MBM rendering procedure 4), 7) . Recently, a specific detection method for porcine DNA using real-time PCR equipment was reported by Tanabe, et al 11) .…”

“…The chosen target sequences for PCR amplification, namely mitochondrial DNA (mtATP8 and mtATP6), have relatively high degrees of variation among vertebrates 7) . Similar sequences are not found in plants 8) .…”