Developmental validation of feline, bovine, equine, and cervid quantitative PCR assays.
Accurate DNA quantification is essential for optimizing DNA testing and minimizing sample consumption. Real-time quantitative polymerase chain reaction (qPCR) assays have been published for human and canine nuclear DNA, and the need for quantifying other forensically important species was evident. Following the strategy employed for the canine qPCR assay, we developed individual assays to accurately quantify feline, bovine, equine, and cervid nuclear DNA. Each TaqMan-based assay incorporates a genus-specific probe targeting the Melanocortin-1 Receptor gene and includes a piece of synthetic DNA that acts as an internal PCR control for detecting inhibition. Developmental validations were carried out following the revised guidelines of the Scientific Working Group on DNA Analysis Methods with modifications necessary for validation of nonhuman qPCR assays. All assays demonstrated the specificity, sensitivity, stability, reproducibility, accuracy, and precision required for forensic casework. The application of these assays to animal forensic DNA analysis has both conserved laboratory resources and improved genotyping results.
University of California, Davis, 95616, USA. cdlindquist@ucdavis.edu
Christina D
CD
Lindquist
Jeffery J
JJ
Evans
Elizabeth J
EJ
Wictum
D000818:Animals; D002415:Cats / Q000235:genetics; D002417:Cattle / Q000235:genetics; D016172:DNA Fingerprinting / Q000592:standards / Q000662:veterinary*; D017931:DNA Primers; D003670:Deer / Q000235:genetics; D004285:Dogs / Q000235:genetics; D005838:Genotype; D006736:Horses / Q000235:genetics; D015345:Oligonucleotide Probes; D016133:Polymerase Chain Reaction / Q000592:standards / Q000662:veterinary*; D044102:Receptor, Melanocortin, Type 1 / Q000235:genetics; D015203:Reproducibility of Results; D013045:Species Specificity; D020080:Tandem Repeat Sequences
D017931:DNA Primers; D015345:Oligonucleotide Probes; D044102:Receptor, Melanocortin, Type 1