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

Abstract

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.

ATP citrate lyase 1 (acl1) gene-based loop-mediated amplification assay for the detection of the Fusarium tricinctum species complex in pure cultures and in cereal samples

“…Only a few applications of LAMP for the detection of fungal organisms have been published to date (Endo et al, 2004;Gadkar and Rillig, 2008;Hayashi et al, 2007;Matsuzawa et al, 2010;Ohori et al, 2006;Sun et al, 2010;Tomlinson et al, 2007;Uemura et al, 2008). Most recently, a LAMP-based detection assay for F. graminearum, the most important producer of the mycotoxins DON and ZEN in cereals and cereal based food and feed and a member of the FHB complex of species was published by authors of the current study (Niessen and Vogel, 2010).…”

Development of a real-time PCR assay for detection of Phytophthora kernoviae and comparison of this method with a conventional culturing technique

“…Nucleic acid-based methods, such as real-time PCR, can reduce diagnosis time to a few hours, and can enable detection in samples from which the pathogen is not culturable (Mumford et al 2006). Such methods also have the advantage that they can be automated for high throughput testing, or adapted for use in the field (Tomlinson et al 2005(Tomlinson et al , 2007Hughes et al 2006a). Nucleic acid-based assays are also compatible with parallel or multiplex testing for other species that occur on the same host, such as P. ramorum (Hughes et al 2006b;Schena et al 2006).…”

Shining a Light on LAMP Assays — A Comparison of LAMP Visualization Methods Including the Novel use of Berberine

“…Because of its inhibitory effect on DNA polymerization at higher concentrations, SYBR Green I is mainly used in LAMP for end point detection by adding it to the reaction mixture after the amplification reaction (13, 14) or by depositing the dye into the lid of the reaction vessel (15). EvaGreen is more applicable for the monitoring of LAMP reactions in real time because it does not affect DNA polymerization and therefore may be present throughout the entire reaction (16, 17). …”