Allen, E.

Anema, J.

Foord, B.

James, D.

Leal, I.

Reisle, C.

Uzunovic, A.

Varga, A.

English

JKI Open Journal Systems (Julius Kühn-Institut)

  IUFRO / REPHRAME International Conference on Pine Wilt Disease 2013  Berichte aus dem Julius Kühn-Institut 169. 2013 39 Leal I, et al., Development of a reverse transcription loop-mediated isothermal amplification (RT-LAMP) method to detect living pinewood nematode, Bursaphelenchus xylophilus, in wood. In: Schröder, T. (ed.), Pine Wilt Disease Conference 2013, pp. 39-40, Braunschweig, ISSN: 1866-590X  Development of a reverse transcription loop-mediated isothermal amplification (RT-LAMP) method to detect living pinewood nematode, Bursaphelenchus xylophilus, in wood. Leal I1 Natural Resources Canada, Canadian Forst Service, 506 W. Burnside Rd. Victoria, BC   V8Z 1M5, Canada 1, Allen E1, Anema J1, Reisle C1, Foord B1, Uzunovic A2, Varga A3, James D3 2 FPInnovations, Forintek Division, 2665 East Mall, Vancouver, BC V6T 1W5, Canada 3 Sidney Laboratory, Centre for Plant Health, Canadian Food and Inspection Agency, 8801 East Saanich Rd., BC V8L 1H3, Canada Email:isabel.leal@nrcan.gc.ca    Current molecular techniques for the detection of PWN rely on the presence of genomic DNA and thus cannot differentiate between living and dead PWN. The detection of dead nematodes could lead to unnecessary trade disruption. We have developed a reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay, which specifically identifies living PWN in wood by detecting the presence of expansin mRNA as a viability marker. This diagnostic method was found to be more sensitive, faster, more cost-effective, and allows for simpler visual detection as compared to PCR. We chose an expansin gene, because it had been sequenced for both PWN and its closest related species, B. mucronatus, (Kikuchi et al., 2009), and because it contains an intron, which is present only in genomic DNA. In order for an RT-LAMP method to be a reliable indicator of viability, it was important to ascertain that only cDNA transcribed from mRNA was amplified, and to eliminate the possibility that any genomic DNA (gDNA) could be amplified.  We designed 6 LAMP primers that recognize 8 distinct regions in the target sequence (Notomi et al., 2000). One of the primers was positioned at an exon-exon junction of the expansin gene, so that genomic DNA could not be amplified. When testing gDNA samples and cDNA from different Bursaphelenchus species, we found exclusive amplification of cDNA from PWN. Positive samples were detected with HNB (hydroxynaphthol blue) by a change of colour from violet to blue (Goto et al., 2009). The sensitivity of the RT-LAMP diagnostic to detect living PWN was higher than that obtained by a conventional PCR diagnostic method, and similar to a real time RT-PCR   IUFRO / REPHRAME International Conference on Pine Wilt Disease 2013  Berichte aus dem Julius Kühn-Institut 169. 2013 40 assay. We modified an RNA extraction protocol (Chomczynski and Sacchi, 1986) in order to improve extraction quality from wood.  We have optimized the RT-LAMP assay not only on nematodes from pure isolate cultures, but also on samples directly isolated from 4 g of PWN-infected wood. This assay was used to test the presence/absence of living PWN in wood that had been heat treated according to ISPM 15 (FAO 2009).  From the results obtained, we found that all heat-treated wood samples were free of living PWN, and thus the heat treatment applied to these wood samples was an effective treatment to kill PWN. We will be using this method to verify the efficacy of other wood treatment such as sulfuryl fluoride and phosphine. This method will help resolve disputes over the detection of PWN by clarifying whether any PWN present in wood is alive or dead. It can also be used to evaluate the efficiency of wood treatment procedures. REFERENCES Kikuchi T; Li H; Karim N; Kennedy M; Moens M; Jones T (2009).  Identification of putative expansin-like genes from the pine wood nematode Bursaphalenchus xylophilus, and evolution of the expansin gene family within the Nematoda.  Nematology 11(3), 355-364. Notomi T; Okayama H; Masubuchi H; Yonekawa T; Watanabe K; Amino N; Hase T (2000). Loop-mediated isothermal amplification of DNA. Nucleic acids research, (12) e63 Goto, M., Honda, E., Ogura, A., Nomoto, A., & Hanaki, K.-I. (2009). Colorimetric detection of loop-mediated isothermal amplification reaction by using hydroxy naphthol blue. BioTechniques, 46(3), 167–72. Chomczynski P; Sacchi N (1986).  Single-Step Method of RNA Isolation by Acid Guanidinium Thiocyanate-Phenol-Chloroform Extraction.  Analytical Biochemistry 162, 156-159. FAO International Standards for Phytosanitary Measures. 2009. Guidelines for regulating wood packaging material in international trade. Publication No. 15. Food and Agriculture Organization, Rome, Italy.    

Development of a reverse transcription loop-mediated isothermal amplification (RT-LAMP) method to detect living pinewood nematode, Bursaphelenchus xylophilus, in wood

https://ojs.openagrar.de/index.php/BerichteJKI/article/download/2559/2809

Development of a reverse transcription loop-mediated isothermal amplification (RT-LAMP) method to detect living pinewood nematode, Bursaphelenchus xylophilus, in wood

Abstract

Similar works

Full text

Available Versions

JKI Open Journal Systems (Julius Kühn-Institut)