Lee et al (1993) developed species specific oligonucleotides from ITS to identify some Phythophthora species by dot blot hybridization. In order to have enough sensitivity, it is necessary to first amplify the DNA from the environmental samples to detect one species. This single species detection approach has been replaced by real-time PCR whereby the species specific hybridization has become the Taqman probe, making the dot blot hybridization obsolete. However, Lévesque et al (1998) demonstrated that the Lee and Taylor hybridization oligonucleotides and some Pythium species they developed could be immobilized in an array format and used in what was called at the time reverse dot blot hybridization. They also demonstrated that immobilized PCR products could not provide species specificity. A diagnostic format where target sequences could be amplified and used to probe a oligonucleotide macro- or microarray for classification of isolates to a species or population level would be especially useful when dealing with species that are under strict regulatory control. A macroarray for the closely related genus Pythium based on ITS sequence differences has been developed for identification of a large number of species (Tambong et al. 2006). A similar effort is currently underway for the development of an array for the identification of Phytophthora species using ITS sequences and several other genetic loci (C.A. Levesque, personal communication); a preliminary report on the ITS based array has been presented (O'Gorman et al. 2000). Lievens et al. (2006) explored the development of an array format capable of detection of SNPs and reported on positioning of base mismatches in the oligonucleotide probe to provide the greatest specificity. In an effort to further improve specificity Anderson et al. (2006) reported on the use of hybridization melting kinetics to help confirm positive results. While the standard array format relies on hybridization between the target sequences and an oligonucleotide probe attached to the array, another format called a padlock probe works on a slightly different principle (Szemes et al. 2005). The terminal regions of the probe are complimentary to adjacent variable sequences capable of differentiating isolates/species. When these terminal regions of the probe anneal to the target sequences it places them adjacent to each other and enables ligase to join them together, thereby circularizing the probe. Forward and reverse primers nested within the terminal species-specific annealing sites are then used to amplify the central portion of the circularized probe in between these primers, which includes a unique sequence that identifies this particular genotype (the unique sequence is custom made to optimize specificity and annealing temperature, thereby making it easier to standardize conditions for multiplex detections).
· Considerations when developing array assays
· Most of the considerations for the development of a PCR assay apply (e.g. quality of DNA, efficient PCR, internal controls, generic probes).
· As discussed in the DNA sequencing section, for identification of a strain DNA sequencing is the best technique and DNA array hybridization would be technical overkill (it would also giving ambiguous results with new species).
· For detection of one or a few species from environmental samples, real time PCR is faster, more quantifiable and more cost effective.
· DNA array hybridization signal correlates well with DNA quantities derived from real time PCR but the latter is considered more accurate (Le Floch, G., Tambong, J., Vallance, J., Tirilly, Y., Lévesque, C.A., and Rey, P. 2007. Rhizosphere persistence of three Pythium oligandrum strains in tomato soilless culture assessed by DNA macroarray and real-time PCR. FEMS Microbiology Ecology 61:317-26.)
· The design of immobilized oligonucleotides is similar to the design of TaqMan probes. They differ from PCR primers by the need to destabilize a hybridization reaction instead of polymerization by Taq polymerase. Mismatches in the middle, especially C/A mismatches, are very good at destabilizing the hybridization.
· All oligonucleotides in a glass or membrane array must hybridize under a single condition and the design of the oligonucleotides must reflect this.
· For additional considerations see
· Seifert, K.A., and Lévesque, C.A. 2004. Phylogeny and molecular diagnosis of mycotoxigenic fungi. European Journal of Plant Pathology 110:449–471.
· Lévesque, C.A. 2007. Molecular diagnostics of soilborne fungal pathogens and applications to disease management. In Biotechnology and Plant Disease Management, edited by Z. K. Punja, S. DeBoer and H. Sanfaçon. Wallingford, UK: CABI Books.
For a review on the use of arrays for pathogen detection see Lievens and Thomma (2005)
Anderson N, Szemes M, O'Brien P, de Weerdt M, Schoen C, Boender P, Bonants P. 2006 Use of hybridization melting kinetics for detecting Phytophthora species using three-dimensional microarrays: demonstration of a novel concept for the differentiation of detection targets. Mycol Res. 110:664-671
O'Gorman, D., de Cock, A.W.A.M. and Lévesque, C.A. (2000) Development of a diagnostic array for pathogenic Phytophthora species. Canadian Journal of Plant Pathology 22:190 (pdf of poster)
Szemes M, Bonants P, de Weerdt M, Baner J, Landegren U, Schoen CD. 2005 Diagnostic application of padlock probes--multiplex detection of plant pathogens using universal microarrays Nucleic Acids Res. 33:-