Developmental validation of feline, bovine, equine, and cervid quantitative PCR assays.
Abstract: 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.
2010 American Academy of Forensic Sciences. Published 2010. This article is a U.S. Government work and is in the public domain in the U.S.A.
Publication Date: 2010-11-11 PubMed ID: 21070239DOI: 10.1111/j.1556-4029.2010.01605.xGoogle Scholar: Lookup
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- Journal Article
- Validation Study
Summary
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This study developed and validated new testing methods to accurately measure DNA from cat, cow, horse, and deer species. These tests have proven to be effective and reliable for forensic analysis and have helped conserve laboratory resources and improve results.
Development of the DNA Quantification Assays
- The researchers used Real-time quantitative polymerase chain reaction (qPCR) assays to target and measure DNA from four species – felines (cats), bovines (cows), equines (horses), and cervids (deer).
- These assays are based on the TaqMan method, which is a commonly used technique for measuring DNA and RNA sequences in a sample.
- Each assay includes a genus-specific probe that targets the Melanocortin-1 Receptor gene, which is present in all four species.
- A synthetic DNA piece is included in each assay, acting as an internal control to check for and measure any possible inhibition (factors preventing the PCR from working correctly).
Validation of the Assays
- The new qPCR assays were thoroughly validated according to the guidelines set by the Scientific Working Group on DNA Analysis Methods.
- Some modifications were made to these guidelines to cater to the specific needs of nonhuman DNA quantification.
- Validation tests included checking the assays for specificity (the ability to accurately target and measure the species-specific DNA), sensitivity (the ability to detect even small amounts of the DNA), stability, reproducibility, accuracy, and precision.
- All assays passed these validation tests and were deemed suitable for use in forensic casework.
Impact of the New Assays
- These new assays offer an accurate method of quantifying DNA from feline, bovine, equine, and cervid species – a capability that was previously lacking in forensic DNA analysis and thus required.
- The new assays have successfully conserved resources in the laboratory by optimizing DNA testing and reducing sample consumption.
- They have also improved genotyping results, potentially enhancing the accuracy and reliability of forensic analysis involving these species.
Cite This Article
APA
Lindquist CD, Evans JJ, Wictum EJ.
(2010).
Developmental validation of feline, bovine, equine, and cervid quantitative PCR assays.
J Forensic Sci, 56 Suppl 1, S29-S35.
https://doi.org/10.1111/j.1556-4029.2010.01605.x Publication
Researcher Affiliations
- University of California, Davis, 95616, USA. cdlindquist@ucdavis.edu
MeSH Terms
- Animals
- Cats / genetics
- Cattle / genetics
- DNA Fingerprinting / standards
- DNA Fingerprinting / veterinary
- DNA Primers
- Deer / genetics
- Dogs / genetics
- Genotype
- Horses / genetics
- Oligonucleotide Probes
- Polymerase Chain Reaction / standards
- Polymerase Chain Reaction / veterinary
- Receptor, Melanocortin, Type 1 / genetics
- Reproducibility of Results
- Species Specificity
- Tandem Repeat Sequences
Citations
This article has been cited 5 times.- Brooks A, Creighton EK, Gandolfi B, Khan R, Grahn RA, Lyons LA. SNP Miniplexes for Individual Identification of Random-Bred Domestic Cats. J Forensic Sci 2016 May;61(3):594-606.
- Ng J, Satkoski J, Premasuthan A, Kanthaswamy S. A nuclear DNA-based species determination and DNA quantification assay for common poultry species. J Food Sci Technol 2014 Dec;51(12):4060-5.
- Du QX, Sun JH, Zhang LY, Liang XH, Guo XJ, Gao CR, Wang YY. Time-dependent expression of SNAT2 mRNA in the contused skeletal muscle of rats: a possible marker for wound age estimation. Forensic Sci Med Pathol 2013 Dec;9(4):528-33.
- Alcoser SY, Kimmel DJ, Borgel SD, Carter JP, Dougherty KM, Hollingshead MG. Real-time PCR-based assay to quantify the relative amount of human and mouse tissue present in tumor xenografts. BMC Biotechnol 2011 Dec 16;11:124.
- Sun JH, Nan LH, Gao CR, Wang YY. Validation of reference genes for estimating wound age in contused rat skeletal muscle by quantitative real-time PCR. Int J Legal Med 2012 Jan;126(1):113-20.
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