A rapid and precise diagnostic method for detecting the Pinewood nematode Bursaphelenchus xylophilus by loop-mediated isothermal amplification.

ABSTRACT Bursaphelenchus xylophilus is the causal agent of pine wilt disease, which is a major forest disease in Japan, Korea, China, Taiwan, and Portugal. A diagnostic method which is rapid, precise, and simple could greatly help the proper management of this disease. Here, we present a novel detection method using loop-mediated isothermal amplification (LAMP) targeting the internal transcribed spacer region of ribosomal DNA of the nematode. Specificity of the primers and LAMP was confirmed using DNA from various nematode species related to B. xylophilus. Our experimental results suggest that LAMP can detect B. xylophilus faster and with higher sensitivity than the traditional diagnostic method. Moreover, because it does not require expensive equipment or specialized techniques, this LAMP-based diagnostic method has the potential to be used under field conditions.

[1]  G. Thorne.  Principles of Nematology , 1962 .

[2]  W. Burgermeister,et al.  First report of Bursaphelenchus xylophilus in Portugal and in Europe , 1999 .

[3]  R. Frederick,et al.  Emerging technologies / Technologies naissantes Real-time PCR and its application for rapid plant disease diagnostics , 2002 .

[4]  Kozaburo Hayashi,et al.  Loop-Mediated Isothermal Amplification for Direct Detection of Mycobacterium tuberculosis Complex, M. avium, and M. intracellulare in Sputum Samples , 2003, Journal of Clinical Microbiology.

[5]  T. Powers.  Nematode molecular diagnostics: from bands to barcodes. , 2004, Annual review of phytopathology.

[6]  S. Lee,et al.  Development of an efficient PCR-based diagnosis protocol for the identification of the pinewood nematode, Bursaphelenchus xylophilus (Nematoda: Aphelenchoididae) , 2004 .

[7]  T. Iwanami,et al.  Characterization of the tufB-secE-nusG-rplKAJL-rpoB Gene Cluster of the Citrus Greening Organism and Detection by Loop-Mediated Isothermal Amplification. , 2005, Plant disease.

[8]  K. Futai,et al.  A nested PCR-based method for detecting the pine wood nematode, Bursaphelenchus xylophilus, from pine wood , 2005 .

[9]  X. Z. Liu,et al.  Detection of the Pinewood Nematode, Bursaphelenchus xylophilus, Using a Real-Time Polymerase Chain Reaction Assay. , 2005, Phytopathology.

[10]  Margaret J. Green,et al.  An effective PCR-based diagnostic method for the detection of Bursaphelenchus xylophilus (Nematoda: Aphelenchoididae) in wood samples from lodgepole pine , 2005 .

[11]  N. Boonham,et al.  Satellite DNA as a target for TaqMan real-time PCR detection of the pinewood nematode, Bursaphelenchus xylophilus. , 2007, Molecular plant pathology.

[12]  S. Subbotin,et al.  Molecular diagnostics of plant-parasitic nematodes. , 2007 .

[13]  John T Jones,et al.  Expressed sequence tag (EST) analysis of the pine wood nematode Bursaphelenchus xylophilus and B. mucronatus. , 2007, Molecular and biochemical parasitology.

[14]  N. Kanzaki.  Taxonomy and Systematics of the Nematode Genus Bursaphelenchus (Nematoda: Parasitaphelenchidae) , 2008 .

[15]  John T Jones,et al.  Bursaphelenchus xylophilus: opportunities in comparative genomics and molecular host-parasite interactions. , 2008, Molecular plant pathology.

[16]  Yasuyoshi Mori,et al.  Loop-mediated isothermal amplification (LAMP) of gene sequences and simple visual detection of products , 2008, Nature Protocols.

[17]  Eun Kyu Lee,et al.  Loop Mediated Isothermal Amplification of DNA , 2008 .

[18]  D. Hunt,et al.  Pine wilt disease: a worldwide threat to forest ecosystems , 2009 .