This project assesses two aspects of DNA detection of Phytophthora species from soil samples. Firstly, a nested PCR protocol was established with both primary and nested PCR specific for P. cinnamomi detection. PCR amplification of P. cinnamomi DNA isolated from soil was optimised with the addition of bovine serum albumin and formamide. This was found to improve both the specificity and sensitivity of PCR amplification of DNA in the presence of inhibitors co-extracted along with the target DNA from soil samples. The application of diagnostic nested PCR with the addition of BSA and formamide was verified by comparison with routine culture based detection methods. In all cases, nested PCR detection incorporating BSA and formamide was found to be considerably more sensitive than the culture based detection methods.
The second component of this thesis investigates the simultaneous detection of multiple species of Phytophthora using microarray analysis. Microarray based detection has been previously limited by variable and inconsistent hybridisation intensities across the diversity of probes used in each array. In this study a novel concept for the differentiation of detection targets using duplex melting kinetics is introduced. A microarray assay was developed on a PamChip ¥ microarray enabling the differentiation of target Phytophthora species using the melting kinetics of probe-target duplexes. In the majority of cases the hybridization kinetics of target and non-target duplexes differed significantly. Analysis of the melting kinetics of duplexes formed by probes with target and non-target DNA was found to be an effective method for determining specific hybridization and was independent of fluctuations in hybridization signal intensity. This form of analysis was more robust than the traditional approach based on hybridisation intensity, and allowed the detection of individual Phytophthora species and mixtures there of.
| Identifer | oai:union.ndltd.org:ADTP/221865 |
| Date | January 2006 |
| Creators | N.Williams@murdoch.edu.au, Nari Michelle Anderson |
| Publisher | Murdoch University |
| Source Sets | Australiasian Digital Theses Program |
| Language | English |
| Detected Language | English |
| Rights | http://www.murdoch.edu.au/goto/CopyrightNotice, Copyright Nari Michelle Anderson |
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