title: Validation of a PCR-based method for the detection of various rendered materials in feedstuffs using a forensic DNA extraction kit.
abstract: 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.
authors.affiliation: US. Food and Drug Administration, Center for Veterinary Medicine, Laurel, Maryland 20708, USA. mmyers@cvm.fda.gov
authors.forename: Michael J
authors.initials: MJ
authors.lastname: Myers
authors.forename: Haile F
authors.initials: HF
authors.lastname: Yancy
authors.forename: Michael
authors.initials: M
authors.lastname: Araneta
authors.forename: Jennifer
authors.initials: J
authors.lastname: Armour
authors.forename: Janice
authors.initials: J
authors.lastname: Derr
authors.forename: Lawrence A D
authors.initials: LA
authors.lastname: Hoostelaere
authors.forename: Doris
authors.initials: D
authors.lastname: Farmer
authors.forename: Falana
authors.initials: F
authors.lastname: Jackson
authors.forename: William M
authors.initials: WM
authors.lastname: Kiessling
authors.forename: Henry
authors.initials: H
authors.lastname: Koch
authors.forename: Huahua
authors.initials: H
authors.lastname: Lin
authors.forename: Yan
authors.initials: Y
authors.lastname: Liu
authors.forename: Gabrielle
authors.initials: G
authors.lastname: Mowlds
authors.forename: David
authors.initials: D
authors.lastname: Pinero
authors.forename: Ken L
authors.initials: KL
authors.lastname: Riter
authors.forename: John
authors.initials: J
authors.lastname: Sedwick
authors.forename: Yuelian
authors.initials: Y
authors.lastname: Shen
authors.forename: June
authors.initials: J
authors.lastname: Wetherington
authors.forename: Ronsha
authors.initials: R
authors.lastname: Younkins
mesh_terms: D000821:Animal Feed / Q000032:analysis*; D000818:Animals; D002417:Cattle; D004247:DNA / Q000032:analysis*; D017931:DNA Primers; D016643:Encephalopathy, Bovine Spongiform / Q000517:prevention & control / Q000635:transmission; D005189:False Positive Reactions; D005506:Food Contamination / Q000032:analysis*; D006801:Humans; D007753:Laboratories / Q000592:standards*; D016133:Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D012756:Sheep; D013045:Species Specificity; D013552:Swine; D013997:Time Factors
chemical_list: D017931:DNA Primers; D004247:DNA