Molecular Detection of 10 of the Most Unwanted Alien Forest Pathogens in Canada Using Real-Time PCR.
Invasive alien tree pathogens can cause significant economic losses as well as large-scale damage to natural ecosystems. Early detection to prevent their establishment and spread is an important approach used by several national plant protection organizations (NPPOs). Molecular detection tools targeting 10 of the most unwanted alien forest pathogens in Canada were developed as part of the TAIGA project (http://taigaforesthealth.com/). Forest pathogens were selected following an independent prioritization. Specific TaqMan real-time PCR detection assays were designed to function under homogeneous conditions so that they may be used in 96- or 384-well plate format arrays for high-throughput testing of large numbers of samples against multiple targets. Assays were validated for 1) specificity, 2) sensitivity, 3) precision, and 4) robustness on environmental samples. All assays were highly specific when evaluated against a panel of pure cultures of target and phylogenetically closely-related species. Sensitivity, evaluated by assessing the limit of detection (with a threshold of 95% of positive samples), was found to be between one and ten target gene region copies. Precision or repeatability of each assay revealed a mean coefficient of variation of 3.4%. All assays successfully allowed detection of target pathogen on positive environmental samples, without any non-specific amplification. These molecular detection tools will allow for rapid and reliable detection of 10 of the most unwanted alien forest pathogens in Canada.
Natural Resources Canada, Canadian Forest Service, Laurentian Forestry Centre, Québec, QC, Canada.
Josyanne
J
Lamarche
Natural Resources Canada, Canadian Forest Service, Laurentian Forestry Centre, Québec, QC, Canada.
Amélie
A
Potvin
Natural Resources Canada, Canadian Forest Service, Laurentian Forestry Centre, Québec, QC, Canada.
Gervais
G
Pelletier
Natural Resources Canada, Canadian Forest Service, Laurentian Forestry Centre, Québec, QC, Canada.
Don
D
Stewart
Department of Forest and Conservation Sciences, Faculty of Forestry, University of British Columbia, Vancouver, BC, Canada.
Nicolas
N
Feau
Department of Forest and Conservation Sciences, Faculty of Forestry, University of British Columbia, Vancouver, BC, Canada.
Dario I O
DI
Alayon
Department of Forest and Conservation Sciences, Faculty of Forestry, University of British Columbia, Vancouver, BC, Canada; FPInnovations, Vancouver, BC, Canada.
Angela L
AL
Dale
FPInnovations, Vancouver, BC, Canada.
Aaron
A
Coelho
FPInnovations, Vancouver, BC, Canada.
Adnan
A
Uzunovic
Canadian Food Inspection Agency, Pathogen Identification Research Laboratory (PIRL), Ottawa, ON, Canada.
Guillaume J
GJ
Bilodeau
Canadian Food Inspection Agency, Pathogen Pathology Laboratory (PPL), Ottawa, ON, Canada.
Stephan C
SC
Brière
Natural Resources Canada, Canadian Forest Service, Laurentian Forestry Centre, Québec, QC, Canada; Department of Forest and Conservation Sciences, Faculty of Forestry, University of British Columbia, Vancouver, BC, Canada.
Richard C
RC
Hamelin
Natural Resources Canada, Canadian Forest Service, Laurentian Forestry Centre, Québec, QC, Canada.
Philippe
P
Tanguay
D002170:Canada; D004271:DNA, Fungal / Q000032:analysis; D065928:Forests; D005658:Fungi / Q000145:classification* / Q000235:genetics / Q000302:isolation & purification*; D016533:Mycological Typing Techniques / Q000379:methods / Q000592:standards; D010802:Phylogeny; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods* / Q000592:standards; D012680:Sensitivity and Specificity; D014197:Trees / Q000382:microbiology*
D004271:DNA, Fungal