From the entomological evidence occurring on and around a corpse it is possible to determine an estimated post-mortem interval (PMI). The critical step in this examination is the accurate identification of specimens collected ensuring the application of appropriate species-specific developmental data. Current molecular techniques in the identification of forensically important Calliphoridae species from the Australian region have been explored and found to be a highly significant and valuable area of research. The cytochrome oxidase genes in the mitochondrial genome have been shown to have sufficient sequence diversity to distinguish forensically relevant Calliphoridae species. In order to target the observed sequence diversity within relevant regions of the nuclear or mitochondrial genomes, sequence specific primer (SSP) pairs are used to target polymorphisms, resulting in the amplification of specific species. This technique has proven to be both a rapid and successful identification tool in the analysis of insect taxa, especially Culicidae. SSP typing is particularly useful, as it requires no subsequent sequencing or restriction with enzymes, both of which require additional time and reagents. The aim of this research was to develop a multiplex SSP reaction for the identification of forensically important Calliphoridae species. Seven SSP pairs preliminarily designed by Harvey (2006) were utilised in the identification of Calliphora dubia, Calliphora albifrontalis, Chrysomya rufifacies, Chrysomya megacephala and Lucilia sericata. Once optimised the SSP pairs were developed into two multiplex PCR reactions. This thesis presents the experiments performed, analysis conducted and results obtained through the development of the multiplex SSP-PCR system. Initial testing of the seven preliminarily designed SSP pairs conveyed non-concordance between expected and observed results. Additional species were continually amplified, even after extensive optimisation attempts, including alternations to annealing temperature, MgCl2 and primer concentration. Of the 7 SSP pairs, 6 were re-designed to improve specificity, whilst one was removed from further testing and replaced with 2 newly designed primer pairs. 15 Continual testing of 8 SSP pairs was conducted, but only 6 could be successfully optimised. Optimisation was limited to alterations to annealing temperature, to allow for potential multiplexing. To confirm the regions and species amplified, sequencing of the PCR products was performed. Though only partial sequences were obtained for most samples the alignment shows the expected region amplified with specific species variations. Using the remaining 6 SSP pairs all species tested were identifiable, allowing for multiplexing potential to be tested. Multiplex PCR is a cost effective and efficient technique that is becoming increasing popular within a wide range of scientific disciplines. To date there has been no recorded use of this technique in relation to either forensic entomology or the analysis of forensically important Calliphoridae species. The 6 SSP pairs were manipulated to produce one successful multiplex PCR system using 3 SSP pairs to identify L. sericata, Ch. rufifacies and Ch. megacephala, and one unsuccessful multiplex PCR that amplified a single SSP pair for the identification of C. dubia and Ch. rufifacies. When both reactions are utilised, it is possible to identify all 5 forensically important Calliphoridae species tested.
|Publication status||Unpublished - 2008|