Phytophthora genus-specific primer pair Two primer pairs have been developed for amplification of a Phytophthora genus-specific amplicon that differ in specificity. · Phy-8b + Phy-10b – This primer pair is used for detection of P. ramorum
Sequence alignment of the genus-specific amplicon used for species-specific primer development - .msf, .nex, .fastA Amplification of various Phytophthora spp. with the Phytophthora genus-specific primers FMPh-8b + FMPh-10b with 2% glycerol in the reaction mixture and an annealing temperature of 66° C with 11 µl of the amplification mixture loaded into each well of a 3% NuSieve 3:1 agarose gel. The molecular size marker is a 100-bp ladder from New England BioLabs. In addition to the Phytophthora spp. noted above the Phytophthora genus-specific primers FMPh-8b + FMPh-10b generated amplicons when the following species were used as a template DNA: P. clandestine, P. humicola, P. ideai, P. iranica, P. katsurae, P. medii, P. melonis, P. inflata, P. primulae, P. richardiae, P. porri, P. quercina, P. tentaculata, or isolates from three of the tentative species groupings described by Brasier et al. (2003)(P. taxon Raspberry, P. taxon Pgchlamydo, or Phytophthora sp. “O” group). Test of amplification of various Pythium spp. with the Phytophthora genus-specific primers FMPh-8b + FMPh-10b with 2% glycerol added to the reaction mixture and an annealing temperature of 66° C. A minimum of 100 ng Pythium total DNA was added to each reaction with 12 µl of the amplification mixture loaded into each well of a 3% NuSieve 3:1 agarose gel. The molecular size marker is a 100-bp ladder from New England BioLabs. Test of amplification of various plant species with the Phytophthora genus-specific primers FMPh-8b + FMPh-10b with 2% glycerol added to the reaction mixture and an annealing temperature of 66° C. 12 µl of the amplification mixture loaded into each well of a 3% NuSieve 3:1 agarose gel. The molecular size marker is a 100-bp ladder from New England BioLabs. This primer pair did not amplify sequences from the following additional plants:
Alternative primer pair for amplification of Phytophthora amplicon · FMPhy-8 + FMPhy-10
This primer pair is more robust than FMPhy-8b + FMPhy-10b under the amplification conditions used and will generate greater amounts of amplified product. One possible reason for this is the higher stringency requirements for FMPhy-8b + FMPhy-10b reduces the amount of product amplification.
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