Copy number of target sequences vs marker sensitivity The ITS region is commonly used as a target for diagnostic markers because there is a large amount of sequence data for many species available in GenBank and it is thought to be present in higher copy number than genes associated with house keeping functions, thereby increasing the sensitivity of the molecular assay. Comparing the results from the three TaqMan real-time PCR assays of Bilodeau et al. (2007) provides an opportunity to evaluate what this means from a standpoint of Ct values obtained. When DNA from the field samples infected with P. ramorum was amplified in these three assays the Ct values for the ITS and elicitin markers were essentially the same with both averaging 3.7 cycles less than the β–tubulin marker. Attempts to clarify the copy number of elicitin and β–tubulin genes in the P. ramorum genome were inconclusive (G.J. Bilodeau, unpublished), but given the anticipated copy number of the rDNA repeats relative to house keeping genes it is possible that greater amplification efficiency for the elicitin and β–tubulin markers contributed to the similar level of sensitivity. Nonetheless, in this case the use of these house keeping genes as a diagnostic marker either had no or limited effect on assay sensitivity when compared to the ITS marker. The number of copies of the rDNA repeat unit present in the genome of a Phytophthora spp. has not been reported, but if this genus is similar to what has been observed with Pythium spp. there may be a variable number of repeats present in different isolates of the same species. This should be experimentally verified before this region is used for assays where the extent of pathogen colonization is quantified. With Pythium, PFGE analysis of a number of species revealed that within a species it can be encoded on a variable number of chromosomal sized bands (Martin 1995a). For example, in P. ultimum or P. sylvaticum the rDNA can be encoded on 2 to 5 chromosomal sized bands ranging in size from 2.2 to approximately 5 Mb. Differences in hybridization intensity of rDNA probes relative to other genes following Southern analysis (Martin 1995b) indicate there might also be variation in copy number among isolates of P. sylvaticum, although this has not been experimentally verified. Judelson and Tooley (2000) developed diagnostic markers for P. infestans based on two regions of the nuclear genome that represented families of high copy number sequences (they had between 12,000 and 14,000 copies per nucleus). This high copy number provided a high level of sensitivity for pathogen detection with a single round of conventional PCR able to detect 10 fg pathogen DNA (with a nested amplification it was 0.1 fg). This level of sensitivity was 100 times more sensitive than a single round amplification using a marker developed from ITS sequences. |
