title: A novel real time PCR assay using melt curve analysis for ivory identification.
abstract: Demand for ivory and expansion of human settlements have resulted in a rapid decline in the number of elephants. Enforcement of local and international laws and regulations requires identification of the species from which any ivory, or ivory products, originated. Further geographical assignment of the dead elephant from which the ivory was taken can assist in forensic investigations. In this study, a real-time PCR assay using melt curve analysis was developed and fully validated for forensic use. The presence or absence of three Elephantidae-specific and elephant species-specific melting peaks was used to identify the elephant species. Using 141 blood and ivory samples from the three extant elephant species, the assay demonstrated very high reproducibility and accuracy. The limit of detection was as low as 0.031ng of input DNA for conventional amplification and 0.002ng for nested amplification. Both DNA concentrations are typically encountered in forensic casework, especially for degraded samples. No cross-reactivity was observed for non-target species. Evaluation of direct amplification and nested amplification demonstrated the assay's flexibility and capability of analyzing low-template DNA samples and aged samples. Additionally, blind trial testing showed the assay's suitability application in real casework. In conclusion, wildlife forensic laboratories could use this novel, quick, and low-cost assay to help combat the continuing poaching crises leading to the collapse of elephant numbers in the wild.
authors.affiliation: Forensic Science Program, Department of Applied Science, Faculty of Science, Prince of Songkla University, Thailand. Electronic address: Thitika.k@psu.ac.th.
authors.forename: Thitika
authors.initials: T
authors.lastname: Kitpipit
authors.affiliation: Wildlife Forensic Science Unit, Department of National Parks, Wildlife and Plant Conservation, Thailand.
authors.forename: Kitichaya
authors.initials: K
authors.lastname: Penchart
authors.affiliation: Wildlife Forensic Science Unit, Department of National Parks, Wildlife and Plant Conservation, Thailand.
authors.forename: Kanita
authors.initials: K
authors.lastname: Ouithavon
authors.affiliation: Department of Biology, Faculty of Science, Prince of Songkla University, Thailand.
authors.forename: Chutamas
authors.initials: C
authors.lastname: Satasook
authors.affiliation: School of Biological Sciences, Flinders University, Adelaide, Australia.
authors.forename: Adrian
authors.initials: A
authors.lastname: Linacre
authors.affiliation: Forensic Science Program, Department of Applied Science, Faculty of Science, Prince of Songkla University, Thailand.
authors.forename: Phuvadol
authors.initials: P
authors.lastname: Thanakiatkrai
mesh_terms: D000818:Animals; D003247:Conservation of Natural Resources; D003415:Crime; D016172:DNA Fingerprinting; D004606:Elephants / Q000235:genetics*; D020641:Polymorphism, Single Nucleotide; D060888:Real-Time Polymerase Chain Reaction; D015203:Reproducibility of Results; D013045:Species Specificity; D044366:Transition Temperature
keywords: Elephant; Identification; Ivory; Melt curve; Real time PCR