Potential alternative plant primers The size of the plant amplicon using the FMPl-2b + FMPl-3b primer pair is very close to the size of the P. ramorum diagnostic band (143 bp vs. 135 bp, respectively), which means the first round amplification has to be sufficiently diluted (at least 1:25) before it can be used in the second round amplification with the P. ramorum-specific primer pair to prevent confusion in the diagnosis of P. ramorum. A second set of primers has been developed that generate an amplicon 700 bp in size to replace FMPl-2b + FMPl-3b.
§ FMPl-6 (dTGGAGTGATGGGCACATGCTT) · Replaces FMPl-2b as the forward primer § Sequence alignment used for primer construction (.msf, .fastA) § Primers were constructed from the mitochondrially encoded cytochrome oxidase I gene of Pisum sativum (X14409) and amplify base 81 to 664. § The amplicon is 584 bp in size, which places it a larger than the Phytophthora genus-specific amplicon § Amplification best at 2 mM Mg, good at 3 and 4 mM, less at 1 mM § This primer pair has not been fully evaluated in the multiplex amplification procedure. Before it can be used the following tests need to be done: · Since it is a larger amplicon the efficiency of amplification will be less than FMPl-2b + FMPl-3b so the primer concentration will likely need to be increased above the 0.1 µM level. · It has amplified sequences form the 12 plant species tested thus far, but needs further evaluation with additional species to ensure it is a good general primer pair for plants. |
