Real-Time PCR Detection of Ruminant DNA

2004 ◽  
Vol 67 (3) ◽  
pp. 550-554 ◽  
Author(s):  
LUIS MENDOZA-ROMERO ◽  
EDWARD L. C. VERKAAR ◽  
PAUL H. SAVELKOUL ◽  
ARNOLD CATSBURG ◽  
HENK J. M. AARTS ◽  
...  
Keyword(s):  
Mitochondrial Dna ◽  
Bovine Spongiform Encephalopathy ◽  
Real Time ◽  
Real Time Pcr ◽  
Spongiform Encephalopathy ◽  
Meat And Bone Meal ◽  
Bone Meal ◽  
Dna Targeting ◽  
Species Origin ◽  
Semiquantitative Method

To control the spread of bovine spongiform encephalopathy, several DNA methods have been described for the detection of the species origin of meat and bone meal. Most of these methods are based on the amplification of a mitochondrial DNA segment. We have developed a semiquantitative method based on real-time PCR for detection of ruminant DNA, targeting an 88-bp segment of the ruminant short interspersed nuclear element Bov-A2. This method is specific for ruminants and is able to detect as little as 10 fg of bovine DNA. Autoclaving decreased the amount of detectable DNA, but positive signals were observed in feeding stuff containing 10% bovine material if this had not been rendered in accordance with the regulations, i.e., heated at 134°C for 3 instead of 20 min.

Detection of Rendered Meat and Bone Meals by PCR Is Dependent on Animal Species of Origin and DNA Extraction Method

2010 ◽  
Vol 73 (6) ◽  
pp. 1090-1096 ◽  
Author(s):  
MICHAEL J. MYERS ◽  
DOROTHY E. FARRELL ◽  
CHRISTINE M. DEAVER ◽  
JACQULINE MASON ◽  
HEIDI L. SWAIM ◽  
...  
Keyword(s):  
Real Time ◽  
Dna Extraction ◽  
Real Time Pcr ◽  
Animal Species ◽  
Animal Feed ◽  
Functional Assay ◽  
Threshold Values ◽  
Meat And Bone Meal ◽  
Bone Meal ◽  
Pcr Method

The capability of eight commercially available DNA extraction kits to extract bovine DNA originating in meat and bone meal from fortified feed was evaluated. Four different batches of bovine meat and bone meal (BMBM) were used for DNA extraction with the eight commercial DNA extraction kits. Within each kit, there were minimal differences in the batch-to-batch amounts of extracted DNA. There were differences between the kits in the amounts of DNA that could be extracted from the same amount of starting BMBM. These differences did not translate into differences in the amount of amplifiable DNA from BMBM-fortified dairy feed. Using a validated real-time PCR method, the kit yielding the highest amount extractable DNA was completely unable to yield a positive PCR result; one other kit was also unable to produce a positive PCR result from DNA extracted from BMBM-fortified feed. There was a complete lack of a correlation between the amount of bovine DNA isolated from BMBM by a given extraction kit compared with the relative amounts of DNA isolated from fortified animal feed as evidenced by the cycle threshold values generated using the real-time PCR method. These results demonstrate that extraction of DNA from processed animal protein is different for pure ingredients and fortified animal feeds. These results indicate that a method specifically developed using just animal-derived meat and bone meal may not yield a functional assay when used to detect animal tissues in complete animal feed.

Are Bacterial Toxins Involved in the Aetiology of Transmissible Spongiform Encephalopathies?

1997 ◽  
Vol 11 (4) ◽  
pp. 301-307 ◽  
Author(s):  
T. Stockdale
Keyword(s):  
Bovine Spongiform Encephalopathy ◽  
Bacterial Toxins ◽  
Transmissible Spongiform Encephalopathies ◽  
Spongiform Encephalopathy ◽  
Meat And Bone Meal ◽  
Bone Meal ◽  
Spongiform Encephalopathies ◽  
Infected Meat ◽  
Disease Epidemics ◽  
The Brain

It is argued that the conditions for Bovine Spongiform Encephalopathy and Creutzfeld-Jacob disease epidemics necessitate, in addition to a diet that contains infected meat and bone meal, the presence of leaky membranes that enable bacterial toxins to circulate in the blood and enter the brain.

Real-Time Polymerase Chain Reaction Detection of Bovine DNA in Meat and Bone Meal Samples

2002 ◽  
Vol 65 (7) ◽  
pp. 1158-1165 ◽  
Author(s):  
S. LAHIFF ◽  
M. GLENNON ◽  
J. LYNG ◽  
T. SMITH ◽  
N. SHILTON ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Meat And Bone Meal ◽  
Bone Meal ◽  
Chain Reaction ◽  
Pcr Assay ◽  
The Real ◽  
Pcr Product ◽  
Pcr Products ◽  
Polymerase Chain

We describe a real-time polymerase chain reaction (PCR) assay for the detection of bovine DNA extracted from meat and bone meal (MBM) samples. PCR primers were used to amplify a 271-bp region of the mitochondrial ATPase 8–ATPase 6 gene, and a fluorogenic probe (BOV1) labeled with a 5′ FAM reporter and a 3′ TAMRA quencher was designed to specifically detect bovine PCR product. The specificity of the BOV1 probe for the detection of the bovine PCR product was confirmed by Southern blot hybridization analysis of the probe with PCR products generated from ovine, porcine, and bovine genomic DNA extracted from blood and with PCR products generated from genomic DNA extracted from single-species laboratory scale rendered MBM samples. The specificity of the BOV1 probe was also evaluated in real-time PCR reactions including these genomic targets. Both methods demonstrated that the BOV1 probe was specific for the detection of bovine PCR product. The BOV1 probe had a detection limit of 0.0001% bovine material by Southern blot DNA probe hybridization analysis and a detection limit of 0.001% bovine material in the real-time PCR assay. Application of the real-time PCR assay to six industrial samples that had previously tested positive for the presence of bovine material with a conventional PCR assay yielded positive results with the real-time PCR assay for four samples.

A quantitative assessment of the risk of exposure to bovine spongiform encephalopathy via meat-and-bone meal in Japan

2006 ◽  
Vol 75 (3-4) ◽  
pp. 221-238 ◽  
Author(s):  
Takehisa Yamamoto ◽  
Toshiyuki Tsutsui ◽  
Takashi Nonaka ◽  
Sota Kobayashi ◽  
Akiko Nishiguchi ◽  
...  
Keyword(s):  
Bovine Spongiform Encephalopathy ◽  
Quantitative Assessment ◽  
Spongiform Encephalopathy ◽  
Meat And Bone Meal ◽  
Bone Meal ◽  
Risk Of Exposure

Development of Primers for Detection of Meat and Bone Meal in Ruminant Feed and Identification of the Animal of Origin

2004 ◽  
Vol 67 (6) ◽  
pp. 1289-1292 ◽  
Author(s):  
T. KUSAMA ◽  
T. NOMURA ◽  
K. KADOWAKI
Keyword(s):  
Bovine Spongiform Encephalopathy ◽  
Atp Synthase ◽  
Individual Species ◽  
Spongiform Encephalopathy ◽  
Target Sequence ◽  
Meat And Bone Meal ◽  
Bone Meal ◽  
Cattle Feed ◽  
Important Prerequisite ◽  
Ruminant Feed

The safe use of cattle feed free from meat and bone meal is an important prerequisite to prevent further spread of bovine spongiform encephalopathy. We designed primers to detect very small amounts of meat and bone meal in ruminant feed. Mitochondrial subunit 8 of the ATP synthase gene was used as a target sequence. PCR-based assays revealed amplification of DNA from mammals, ruminants, and individual species using these primers. The method allowed detection of the presence of meat and bone meal in ruminant feed from 0.1 to 0.01%. Sensitivity and effectiveness of the method for detecting prohibited animal proteins in ruminant feed was evaluated.

scholarly journals Atypical H-Type Bovine Spongiform Encephalopathy in a Cow Born after the Reinforced Feed Ban on Meat-and-Bone Meal in Europe

2012 ◽  
Vol 50 (12) ◽  
pp. 4171-4174 ◽  
Author(s):  
C. Guldimann ◽  
M. Gsponer ◽  
C. Drogemuller ◽  
A. Oevermann ◽  
T. Seuberlich
Keyword(s):  
Bovine Spongiform Encephalopathy ◽  
Spongiform Encephalopathy ◽  
Meat And Bone Meal ◽  
Bone Meal

Novel approach for interlaboratory transfer of real-time PCR methods: detecting bovine meat and bone meal in feed

2009 ◽  
Vol 394 (5) ◽  
pp. 1423-1431 ◽  
Author(s):  
Marta Prado ◽  
Olivier Fumière ◽  
Ana Boix ◽  
Aline Marien ◽  
Gilbert Berben ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Meat And Bone Meal ◽  
Bone Meal ◽  
Novel Approach

scholarly journals Real-Time Polymerase Chain Reaction Approach for Quantitation of Ruminant-Specific DNA to Indicate a Correlation Between DNA Amount and Meat and Bone Meal Heat Treatments

2005 ◽  
Vol 88 (5) ◽  
pp. 1399-1403 ◽  
Author(s):  
Barbara Chiappini ◽  
Gianfranco Brambilla ◽  
Umberto Agrimi ◽  
Gabriele Vaccari ◽  
Henk J M Aarts ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Real Time ◽  
False Negative ◽  
The United States ◽  
Spongiform Encephalopathy ◽  
Meat And Bone Meal ◽  
Bone Meal ◽  
Chain Reaction ◽  
Dna Amount ◽  
Polymerase Chain

Abstract The use of ruminant-derived proteins in ruminant feeds has been banned in both the European Union and the United States to prevent further spread of bovine spongiform encephalopathy. Enforcement of these regulations relies on the ability to identify the presence of prohibited proteins in feed. We developed a quantitative real-time polymerase chain reaction assay for the quantification of ruminant-specific DNA as index of protein content. The assay is based on the amplification of a 117 base pair mitochondrial 16S rRNA DNA gene fragment and an internal positive control (IPC). The use of an IPC permits compensation for differences in DNA extraction efficiency and avoids the occurrence of false-negative results. We demonstrated a decrease in target DNA amount with a difference of 2 logs between meat and bone meal (MBM) treated at 133° and 145°C. Such a difference indicates that bias could occur when DNA-based methods are used for quantitation purposes. Risk management could benefit from future efforts concerning validation of the method for MBM detection in feedstuff and safety evaluation of the use of animal-derived proteins in animal nutrition.

Real-Time PCR Detection and Identification of Prohibited Mammalian and Avian Material in Animal Feeds

2009 ◽  
Vol 72 (5) ◽  
pp. 1055-1062 ◽  
Author(s):  
SAIRA CAWTHRAW ◽  
GINNY C. SAUNDERS ◽  
TREVOR C. MARTIN ◽  
JASON SAWYER ◽  
OTTO WINDL ◽  
...  
Keyword(s):  
16S Rrna ◽  
Real Time ◽  
Real Time Pcr ◽  
Animal Feed ◽  
Meat And Bone Meal ◽  
Bone Meal ◽  
Detection And Identification ◽  
Pcr Assays ◽  
Species Specific ◽  
Feed Samples

A method for the detection and identification of “prohibited” mammalian or avian material in animal feed was developed and assessed through the analysis of DNA. A generic real-time PCR assay was designed to detect the presence of mammalian and avian mitochondrial DNA 16S rRNA genes in animal feed samples. Samples positive with this screening method were further investigated using identification assays to detect the 16S rRNA gene from bovine, ovine, porcine, and avian species and to determine whether the DNA originated from species whose material is prohibited from inclusion in farmed animal feed. An internal positive control was coamplified in the 16S real-time PCR assays to monitor PCR amplification efficiency and avoid potential false-negative results. Using vegetable-based feed standards spiked with meat and bone meal generated with a commercial rendering process, 0.1% meat and bone meal could be detected using the general and species-specific 16S assays. The species-specific assays had 100% specificity for the homologous target species. The 16S real-time PCR assays were evaluated alongside existing tests based on protein evaluation or microscopic examination for a wide range of commercial animal feed samples. In total, 111 (0.76%) of 14,678 samples examined contained prohibited material based on the results from at least one of these tests. However, most positive results did not represent noncompliance because they were associated with samples of pet food, which can legitimately contain material prohibited for use in food for farmed animals. The species-specific 16S assays confirmed the presence of prohibited material in 75% of the 111 samples, whereas the existing protein and microscope tests confirmed the presence of this material in 25 and 54% of the samples, respectively.

PCR Detection of Bovine Mitochondrial DNA Derived from Meat and Bone Meal in Feed

2004 ◽  
Vol 67 (12) ◽  
pp. 2829-2832 ◽  
Author(s):  
ATSUSHI TOYODA ◽  
MITSURU NAKAJO ◽  
HIROYUKI KAWACHI ◽  
TOHRU MATSUI ◽  
HIDEO YANO
Keyword(s):  
Mitochondrial Dna ◽  
Sodium Hypochlorite ◽  
Animal Feed ◽  
Milk Replacer ◽  
Spongiform Encephalopathy ◽  
Sodium Hypochlorite Solution ◽  
Selective Detection ◽  
Meat And Bone Meal ◽  
Bone Meal ◽  
Bone Particles

Because bovine meat and bone meal (MBM) is thought to be a major source of bovine spongiform encephalopathy, we developed a PCR-based method for detection of bovine MBM in animal feed. We isolated bone particles from feed containing bovine MBM using a separation technique based on specific gravity and then washed bone particles with sodium hypochlorite solution and an EDTA–proteinase K solution. The mitochondrial DNA was extracted from bone particles and amplified using PCR with cattle-specific primers. Bovine DNA was not detected in a milk replacer containing dried skim milk and dried whey, but bovine DNA was detected in the milk replacer that was mixed with bovine MBM. Other cattle-derived materials in feeds did not interfere with the selective detection of bovine MBM. This method allowed detection of bovine mitochondrial DNA in feed with 0.1% added bovine MBM. When the treatment with sodium hypochlorite was excluded, bovine DNA derived from MBM could not be distinguished from bovine DNA derived from other bovine materials. However, the exclusion of this treatment improved the detection limit of bovine MBM in feed. This method appears suitable for the selective detection of bovine MBM in feed.

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