Molecular Detection of <i>Phytophthora ramorum</i> by Real-Time Polymerase Chain Reaction Using TaqMan, SYBR Green, and Molecular Beacons

Bilodeau, Guillaume J.; Lévesque, C. André; Cock, A. W. A. M. de; Duchaine, Caroline; Brière, S. C.; Uribe, Pedro; Martin, Frank N.; Hamelin, Richard

doi:10.1094/phyto-97-5-0632

Cited by 93 publications

(67 citation statements)

References 54 publications

(72 reference statements)

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“…Improved specificity would allow undiluted extracts from cultures to be tested without the risk of false-positive results. One approach to improving the discrimination of similar species is to design a new assay for a different region of the genome (2,13,29). However, modifications to existing assays can also be made to improve specificity without the need to completely redesign the primers and/or probe.…”

mentioning

confidence: 99%

Faster, Simpler, More-Specific Methods for Improved Molecular Detection of Phytophthora ramorum in the Field

Tomlinson¹,

Barker²,

Boonham³

2007

Appl Environ Microbiol

138

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Phytophthora ramorum is the causal agent of sudden oak death. The pathogen also affects a wide range of tree, shrub, and herbaceous species in natural and landscaped environments as well as plants in the nursery industry. A TaqMan real-time PCR method for the detection of this pathogen in the field has been described previously; this paper describes the development of a number of assays based on this method which have various advantages for use in the field. A scorpion real-time PCR assay that is twice as fast as TaqMan was developed, allowing the detection of P. ramorum in less than 30 min. Also designed was a loop-mediated isothermal amplification (LAMP) assay, which allowed sensitive and specific detection of P. ramorum in 45 min using only a heated block. A positive reaction was identified by the detection of the LAMP product by color change visible to the naked eye.

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mentioning

confidence: 99%

Faster, Simpler, More-Specific Methods for Improved Molecular Detection of Phytophthora ramorum in the Field

Tomlinson¹,

Barker²,

Boonham³

2007

Appl Environ Microbiol

138

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“…In this study, the sensitivity of Molecular beacon and TaqMan probes against a dilution series of P. fragariae genomic DNA was equivalent (Bonants et al 2004). Also, Bilodeau et al (2007) compared three different chemistries including SYBR Green, TaqMan, and Molecular beacons using sequences of β-tubulin, ITS and elicitin gene regions for detection of P. ramorum causing sudden oak death. The results showed that all three real-time PCR assays could separate the pathogen from 65 other species of Phytophthora in all infected samples.…”

Section: Detection Of Plant Pathogensmentioning

confidence: 99%

Real-time PCR applied to study on plant pathogens: potential applications in diagnosis - a review

Mirmajlessi¹,

Loit²,

Mänd³

et al. 2015

Plant Prot. Sci.

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Mirmajlessi S.M., Loit E., Mänd M., Mansouripour S.M. (2015): Real-time PCR applied to study on plant pathogens: potential applications in diagnosis -a review. Plant Protect Sci., 51: 177-190.Quantitative real-time PCR (qPCR) technique incorporates traditional polymerase chain reaction (PCR) efficiency with the production of a specific fluorescent signal, measuring the kinetics of the reaction in the early PCR phases and providing quantification of specific targets in various environmental samples. There are an increasing number of chemistries to detect PCR products, which are widely used in plant pathology as they cluster into the amplicon sequence non-specific and sequence-specific techniques. In this review, we illustrate a general description of major chemistries and discuss some considerations for assay development as it applies for a wide range of applications in epidemiological studies. The technique has become the gold standard for early detection of pathogens and a fundamental tool in the research laboratory.

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“…Many PCR assays were developed for P. ramorum (e.g. Tomlinson et al 2007;Bilodeau et al 2007;Tooley et al 2006;Martin et al 2004;Hughes et al 2006;Hayden et al 2006), to the point of causing some confusion in the international regulatory community as to which one should be routinely used. The international ring trial to evaluate several of these methods simultaneously with the same samples should become a model for other pathogens ).…”

Section: Diagnostics and Molecular Detectionmentioning

confidence: 99%

Fifty years of oomycetes—from consolidation to evolutionary and genomic exploration

Lévesque

2011

Fungal Diversity

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Transformative changes in biological sciences during the past 25 years have led to many significant advances in oomycete research. Before the last half century there were some hints that the oomycetes were related to some algae but it is now definitively demonstrated that they do not share an evolutionary path with kingdom Eumycota and are instead placed in a new kingdom Straminipila. Clarifying this once and for all has created many opportunities, but the rapid expansion of the research community has caused some fragmentation, probably much more so than in other groups of fungi because of a lack of a unifying forum for the members of the community working on issues such as taxonomy or phylogeny. Prior to the advent of molecular phylogenetics, mycologists working in zoosporic fungi were examining the ultrastructure of the zoospore, mainly focussing on the flagellar apparatus, and managed to generate phylogenies or define clades of zoospore producing fungi that remained for the most part valid after the advances in molecular biology. Comprehensive molecular phylogenies that have been published for some genera of the oomycetes have helped in recognising a large number of new species and in the development of a wide range of DNA-based diagnostic tools. The number of genomes available for this group is increasing rapidly, pushing further the discoveries of novel host-parasite interaction mechanisms in oomycetes. Some important plant diseases that were believed to be under control have re-emerged and many new diseases have appeared particularly in forestry and even in mammals. The research community has been able to respond rapidly and effectively to these new challenges. New ecological roles for the oomycetes were found in the suppression of plant diseases and reduction of plant invasineness in natural ecosystems. There are still many challenges ahead in the oomycete community, probably the most pressing one is to establish a robust tree of life foundation like the Assembling the Fungal Tree of Life initiative. The oomycete research community is dynamic and has put to very good use the many new technological advances.

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Molecular Detection of Phytophthora ramorum by Real-Time Polymerase Chain Reaction Using TaqMan, SYBR Green, and Molecular Beacons

Cited by 93 publications

References 54 publications

Faster, Simpler, More-Specific Methods for Improved Molecular Detection of Phytophthora ramorum in the Field

Faster, Simpler, More-Specific Methods for Improved Molecular Detection of Phytophthora ramorum in the Field

Real-time PCR applied to study on plant pathogens: potential applications in diagnosis - a review

Fifty years of oomycetes—from consolidation to evolutionary and genomic exploration

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