Species-specific diagnostic markers   

When the objective is to determine if a particular species is present in a plant sample, species-specific diagnostic markers are needed. These primers should not amplify plants or other organisms that may be present, including closely related Phytophthora species. A variety of sequences have been used for development of these diagnostic markers for a number of species in the genus, including random clones and SCARs (Table 1) as well as individual genes (Table 2). Species-specific diagnostic marker systems for detection of multiple species have been designed from particular regions of the genome. For additional details (including the sequence alignments used for primer development) see:

· ITS region
· Ypt1 gene
· Elicitin and β-tubulin
· Cox spacer region

In addition to conventional and real-time PCR there are several other molecular techniques that have been reported to be useful in molecular diagnostics. Tooley et l. (2002) reported on the use of ligase chain reaction to join two adjacent 24 bp primers that had annealed to the target site in the ITS region of P. infestans. The resulting 48 bp fragment was diagnostic for P. infestans and the related species P. mirabilis and P. phaseoli. The specificity of the assay was greater than previously described PCR primers however the sensitivity was reduced to a limit of detection on a polyacrylamide gel of 0.1 ng. The sensitivity of detection was increased by using a primer pair external to the ligase chain reaction primers as PCR primers and amplifying a larger region in a first round PCR prior to the ligase chain reaction. The sensitivity was also increased by using biotin and digoxygenin labeled primers in the ligase chain reaction and concentrating the template by using streptavidin linked paramagnetic particles prior to using an ELISA assay for digoxigenin qantification. Tomlinson et al. (2007) reported on using loop-mediated isothermal amplification (LAMP) for detection of P. ramorum. One of the advantages of this technique is it doesn't require sophisticated equipment like a thermal cycler to conduct the assay or collect the data, however, the sensitivity of detection is less that PCR (10 pg target DNA).

· Considerations when developing species-specific PCR diagnostic assays
· Discussion on target copy number vs marker sensitivity

Tomlinson JA, Barker I, Boonham N. 2007 Faster, simpler, more-specific methods for improved molecular detection of Phytophthora ramorum in the field. Applied and environmental microbiology. 73:4040-4047

P.W. Tooley, E. Hatziloukas, D.L. Scott, and M.M. Carras 2002 Use of ligase chain reaction for enhanced detection of Phytophthora infestans Canadian Journal of Plant Pathology 24:294-301