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.

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.

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.

Validation of a PCR-Based Method for the Detection of Various Rendered Materials in Feedstuffs Using a Forensic DNA Extraction Kit

2006 ◽  
Vol 69 (1) ◽  
pp. 205-210 ◽  
Author(s):  
MICHAEL J. MYERS ◽  
HAILE F. YANCY ◽  
MICHAEL ARANETA ◽  
JENNIFER ARMOUR ◽  
JANICE DERR ◽  
...  
Keyword(s):  
False Positive ◽  
Animal Feed ◽  
Limit Of Detection ◽  
False Positive Rate ◽  
Meat And Bone Meal ◽  
Bone Meal ◽  
Average Accuracy ◽  
Positive Rate ◽  
Feed Samples ◽  
Pcr Primer

A method trial was initiated to validate the use of a commercial DNA forensic kit to extract DNA from animal feed as part of a PCR-based method. Four different PCR primer pairs (one bovine pair, one porcine pair, one ovine primer pair, and one multispecies pair) were also evaluated. Each laboratory was required to analyze a total of 120 dairy feed samples either not fortified (control, true negative) or fortified with bovine meat and bone meal, porcine meat and bone meal (PMBM), or lamb meal. Feeds were fortified with the animal meals at a concentration of 0.1% (wt/wt). Ten laboratories participated in this trial, and each laboratory was required to evaluate two different primer pairs, i.e., each PCR primer pair was evaluated by five different laboratories. The method was considered to be validated for a given animal source when three or more laboratories achieved at least 97% accuracy (29 correct of 30 samples for 96.7% accuracy, rounded up to 97%) in detecting the fortified samples for that source. Using this criterion, the method was validated for the bovine primer because three laboratories met the criterion, with an average accuracy of 98.9%. The average false-positive rate was 3.0% in these laboratories. A fourth laboratory was 80% accurate in identifying the samples fortified with bovine meat and bone meal. A fifth laboratory was not able to consistently extract the DNA from the feed samples and did not achieve the criterion for accuracy for either the bovine or multispecies PCR primers. For the porcine primers, the method was validated, with four laboratories meeting the criterion for accuracy with an average accuracy of 99.2%. The fifth laboratory had a 93.3% accuracy outcome for the porcine primer. Collectively, these five laboratories had a 1.3% false-positive rate for the porcine primer. No laboratory was able to meet the criterion for accuracy with the ovine primers, most likely because of problems with the synthesis of the primer pair; none of the positive control DNA samples could be detected with the ovine primers. The multispecies primer pair was validated in three laboratories for use with bovine meat and bone meal and lamb meal but not with PMBM. The three laboratories had an average accuracy of 98.9% for bovine meat and bone meal, 97.8% for lamb meal, and 63.3% for PMBM. When examined on an individual laboratory basis, one of these four laboratories could not identify a single feed sample containing PMBM by using the multispecies primer, whereas the other laboratory identified only one PMBM-fortified sample, suggesting that the limit of detection for PMBM with this primer pair is around 0.1% (wt/wt). The results of this study demonstrated that the DNA forensic kit can be used to extract DNA from animal feed, which can then be used for PCR analysis to detect animal-derived protein present in the feed sample.

Application of Quantitative Real-Time PCR in the Detection of Prion-Protein Gene Species-Specific DNA Sequences in Animal Meals and Feedstuffs

2006 ◽  
Vol 69 (4) ◽  
pp. 891-896 ◽  
Author(s):  
FEDERICA BELLAGAMBA ◽  
SERGIO COMINCINI ◽  
LUCA FERRETTI ◽  
FRANCO VALFRÈ ◽  
VITTORIO M. MORETTI
Keyword(s):  
Real Time ◽  
Prion Protein ◽  
Dna Sequences ◽  
Real Time Pcr ◽  
Animal Feed ◽  
Quantitative Estimation ◽  
Taqman Assay ◽  
Quantitative Real Time Pcr ◽  
The Real ◽  
Species Specific

This study describes a method for quantitative and species-specific detection of animal DNA from different species (cattle, sheep, goat, swine, and chicken) in animal feed and feed ingredients, including fish meals. A quantitative real-time PCR approach was carried out to characterize species-specific sequences based on the amplification of prion-protein sequence. Prion-protein species-specific primers and TaqMan probes were designed, and amplification protocols were optimized in order to discriminate the different species with short PCR amplicons. The real-time quantitative PCR approach was also compared to conventional species-specific PCR assays. The real-time quantitative assay allowed the detection of 10 pg of ruminant, swine, and poultry DNA extracted from meat samples processed at 130°C for 40 min, 200 kPa. The origin of analyzed animal meals was characterized by the quantitative estimation of ruminant, swine, and poultry DNA. The TaqMan assay was used to quantify ruminant DNA in feedstuffs with 0.1% of meat and bone meal. In conclusion, the proposed molecular approach allowed the detection of species-specific DNA in animal meals and feedstuffs.

FAST DETECTION AND IDENTIFICATION OF ICU-RELEVANT BACTERIA BY REAL-TIME PCR AND SPECIES-SPECIFIC PROBES

Shock ◽  
2004 ◽  
Vol 21 (Supplement) ◽  
pp. 107
Author(s):  
S. Klaschik ◽  
L E Lehmann ◽  
A. Hoeft ◽  
F. Stuber
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Fast Detection ◽  
Detection And Identification ◽  
Species Specific

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.

O serogroup specific real time PCR assays for the detection and identification of nine clinically relevant non-O157 STECs

2011 ◽  
Vol 28 (3) ◽  
pp. 478-483 ◽  
Author(s):  
Andrew Lin ◽  
Omar Sultan ◽  
Henry K. Lau ◽  
Evelyn Wong ◽  
Gary Hartman ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Detection And Identification ◽  
Pcr Assays
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