Molecular Detection of 10 of the Most Unwanted Alien Forest Pathogens in Canada Using Real-Time PCR

Josyanne Lamarche; Amélie Potvin; Gervais Pelletier; Don Stewart; Nicolas Feau; Dario I O Alayon; Angela L Dale; Aaron Coelho; Adnan Uzunovic; Guillaume J Bilodeau; Stephan C Brière; Richard C Hamelin; Philippe Tanguay

doi:10.1371/journal.pone.0134265

Molecular Detection of 10 of the Most Unwanted Alien Forest Pathogens in Canada Using Real-Time PCR

PLoS One. 2015 Aug 14;10(8):e0134265. doi: 10.1371/journal.pone.0134265. eCollection 2015.

Affiliations

¹ Natural Resources Canada, Canadian Forest Service, Laurentian Forestry Centre, Québec, QC, Canada.
² Department of Forest and Conservation Sciences, Faculty of Forestry, University of British Columbia, Vancouver, BC, Canada.
³ Department of Forest and Conservation Sciences, Faculty of Forestry, University of British Columbia, Vancouver, BC, Canada; FPInnovations, Vancouver, BC, Canada.
⁴ FPInnovations, Vancouver, BC, Canada.
⁵ Canadian Food Inspection Agency, Pathogen Identification Research Laboratory (PIRL), Ottawa, ON, Canada.
⁶ Canadian Food Inspection Agency, Pathogen Pathology Laboratory (PPL), Ottawa, ON, Canada.
⁷ 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.

Abstract

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.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Canada
DNA, Fungal / analysis
Forests
Fungi / classification*
Fungi / genetics
Fungi / isolation & purification*
Mycological Typing Techniques / methods
Mycological Typing Techniques / standards
Phylogeny
Real-Time Polymerase Chain Reaction / methods*
Real-Time Polymerase Chain Reaction / standards
Sensitivity and Specificity
Trees / microbiology*

Substances

DNA, Fungal

Grants and funding

This work was supported by grants from Genome Canada and Genome British Columbia (Large-Scale Applied Research Program, Grant #164) as well as by the Genomics Research and Development Initiative (GRDI) of Natural Resources Canada, FPInnovations and its government and industry members, and the Canadian Food and Inspection Agency (CFIA).