Sequence alignment used for primer construction (.html 1.7MB, .msf, .fastA)
Amplification of various plant species with the plant primers FMPl-2b + FMPl-3b using an annealing temperature of 66° C with 12 µl of the amplification mixture loaded into each well of a 1.5% agarose gel. The molecular size marker is a 100-bp ladder from New England BioLabs with the size of the plant amplicon 143 bp.
This primer pair also has been found to amplify sequences from the following additional plants:
Acer macrophyllum Heteromeles arbutifolia
Aesculus californica Hibiscus rosa-sinensis
Arbutus menziesii Ligustrum japonica 'Korean choice'
Arbutus unedo - 'Compacta' Liriope sp.
Aucuba japonica - 'variegata' Lonicera sp. 'Hummingbird's gold’
Avacado – ‘Fuerta’ Malus sylvestris – ‘Granny Smith’
Azalea - Geovie Tabor Pink Patio Manzanita sp
Azalea - satsuki 'Getsutoku' Prunus persica
Azalea - satsuki 'Juko' Prunus sp. (plum)
Azalea - satsuki 'Kinpai' Pyrus commumis
Azalea - satsuki 'Nuccio's Wild Cherry' Pseudotsuga menziesii
Azalea - 'Girard's Fuchsia' Rhamnus sp.
Azalea - 'Herbert' Rhaphiolepis indica – ‘Ballerina’
Azalea - 'Hino‑Crimson' Rhododendron sp.
Azalea - 'Holland Red' Salal sp.
Begonia - Tubor hybrid Sambucus sp.
Camelia sp. Umbellularia californica
Camelia - 'Winter's Rose' Verbina - romania mix
Camelia - 'Winter's Star' Viburnum burkwoodii
Camelia - 'Betty Sette' Viburnum opulus - 'Sterile'
Camelia japonica - 'April remembered' Viburnum plicatum tomentosum
Camelia japonica – ‘Fumasaka’ Viburnum rhytidophylloides - 'Allegheny'
Clematis sp. - 'Ramona' Viburnum tinus - 'Compactum'
Cotoneaster apiculata - 'Tom Thumb' Vitis vinifera
Delphinium elatum – ‘Summer Skies’
Euonymus japonica - 'Chollipo'
Hedera helix - 'Thorndale'
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
· From base 81-101 of the cox I gene
§ 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.· It needs to be more thoroughly tested in multiplex amplifications to ensure it does not interfere with the Phytophthora genus-specific amplification.