Validation of a Polymerase Chain Reaction Method for the Detection of Rendered Bovine-Derived Materials in Feedstuffs

Myers, Michael J.; Friedman, Sharon; Farrell, Dorothy E.; Dove-Pettit, Denise A.; Bucker, Michael F.; Kelly, Simon P.; Madzo, Steve; Campbell, Warren L.; Wang, Rongfu; Paine, Donald D.; Cerniglia, Carl E.

doi:10.4315/0362-028x-64.4.564

Cited by 40 publications

(20 citation statements)

References 8 publications

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“…Recently the same team organised a validation study with four laboratories, co-ordinated by the United States Food and Drug Administration (63). In this study, they used the Tartaglia method, even for the extraction protocol, with slight modifications regarding the lysis buffer.…”

Section: Molecular Genetic Methodsmentioning

confidence: 99%

An overview of tests for animal tissues in feeds applied in response to public health concerns regarding bovine spongiform encephalopathy

Gizzi

Raamsdonk²,

Baeten³

et al. 2003

Rev. Sci. Tech. OIE

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Section: Molecular Genetic Methodsmentioning

confidence: 99%

An overview of tests for animal tissues in feeds applied in response to public health concerns regarding bovine spongiform encephalopathy

Gizzi

Raamsdonk²,

Baeten³

et al. 2003

Rev. Sci. Tech. OIE

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“…Indeed this was expected, since swine and wild boar are a same species and cannot be distinguished through the analysis of mitochondrial DNA (Larson et al, 2005). 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).…”

Section: Discussionmentioning

confidence: 99%

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

Frezza¹,

Giambra²,

Chegdani³

et al. 2008

Innovative Food Science & Emerging Technologies

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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.

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“…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 for the presence of animal constituents in ruminant feeds have been proposed and applied (1,16,17,(19)(20)(21)(22)28). Currently, the method of cially recommended by the EU for the control of MBM in animal feeds is the microscopic method, which involves the identi cation of an animal species through analysis of the tissue constituents (mainly bone fragments) on the basis of their morphological characteristics (7).…”

mentioning

confidence: 99%

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

Frezza

Favaro

Vaccari

et al. 2003

Journal of Food Protection

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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...

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Validation of a Polymerase Chain Reaction Method for the Detection of Rendered Bovine-Derived Materials in Feedstuffs

Cited by 40 publications

References 8 publications

An overview of tests for animal tissues in feeds applied in response to public health concerns regarding bovine spongiform encephalopathy

An overview of tests for animal tissues in feeds applied in response to public health concerns regarding bovine spongiform encephalopathy

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

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

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