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The development of a real-time PCR multiplex TaqMan™ assay for vertebrate class in non-human DNA samples for forensic science

Whiteman, Erin Tuxford (2021) The development of a real-time PCR multiplex TaqMan™ assay for vertebrate class in non-human DNA samples for forensic science. Masters by Research thesis, Murdoch University.

PDF - Whole Thesis
Embargoed until May 2023.


Non-human DNA (e.g. animal DNA) has become an increasingly useful tool to forensic science, aiding in linking suspects, victims, and crime scenes (24). However, due to many complexities (eg. the potential for non-human DNA to be from any species and the challenges of distinguishing DNA mixtures) and limited resources, this kind of DNA often remains unanalysed in investigations. Presumptive identification of blood is not always human specific and current DNA testing usually stops when DNA samples fail human DNA testing protocols. An efficient way to routinely detect and confirm the presence of animal DNA within samples is therefore needed. This research outlines the framework and development of a universal Vertebrate Class assay for the detection of Mammalian (mammal), Aves (bird), Fish, and Reptilia (reptile) DNA. The assay functions as a real-time PCR TaqMan multiplex assay, with an internal positive control. Primers and probes have been designed with Geneious Prime, targeting the mitochondrial genome, specifically regions of the cytochrome b gene (Cyt b) and cytochrome c oxidise subunit 1 gene (COX1) (4, 25-29). Multistage testing was conducted (eg. primer and probe screening and selection, SYBR green testing and TaqMan multiplex testing). Varied success was reported for the identification of Mammalian, Aves and Fish DNA samples across all multistage laboratory testing processes. The Reptilia probe was not able to be tested at this time due to unforeseen delays. With necessary further testing and validation, this will be the first Vertebrate Class assay design with potential capabilities of detecting Mammalian, Aves, Fish, and Reptilia classes simultaneously, providing both universal and specific detection in one convenient and rapid assay.

Item Type: Thesis (Masters by Research)
Murdoch Affiliation(s): Medical, Molecular and Forensic Sciences
Notes: Accelerated Research Masters with Training
Supervisor(s): Tobe, Shane, Magni, Paola and Coghlan, Megan
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