title: Satellite DNA as a target for TaqMan real-time PCR detection of the pinewood nematode, Bursaphelenchus xylophilus.
abstract: SUMMARY The pinewood nematode (PWN), Bursaphelenchus xylophilus, is a major pathogen of conifers, which impacts on forest health, natural ecosystem stability and international trade. As a consequence, it has been listed as a quarantine organism in Europe. A real-time PCR approach based on TaqMan chemistry was developed to detect this organism. Specific probe and primers were designed based on the sequence of the MspI satellite DNA family previously characterized in the genome of the nematode. The method proved to be specific in tests with target DNA from PWN isolates from worldwide origin. From a practical point of view, detection limit was 1 pg of target DNA or one individual nematode. In addition, PWN genomic DNA or single individuals were positively detected in mixed samples in which B. xylophilius was associated with the closely related non-pathogenic species B. mucronatus, up to the limit of 0.01% or 1% of the mixture, respectively. The real-time PCR assay was also used in conjunction with a simple DNA extraction method to detect PWN directly in artificially infested wood samples. These results demonstrate the potential of this assay to provide rapid, accurate and sensitive molecular identification of the PWN in relation to pest risk assessment in the field and quarantine regulation.
authors.affiliation: INRA, UMR1064 Interactions Plantes-Microorganismes et Santé Végétale, 400 route des Chappes, BP167, F-06903 Sophia Antipolis, France.
authors.forename: Cecile
authors.initials: C
authors.lastname: François
authors.forename: Chantal
authors.initials: C
authors.lastname: Castagnone
authors.forename: Neil
authors.initials: N
authors.lastname: Boonham
authors.forename: Jenny
authors.initials: J
authors.lastname: Tomlinson
authors.forename: Rebecca
authors.initials: R
authors.lastname: Lawson
authors.forename: Sue
authors.initials: S
authors.lastname: Hockland
authors.forename: James
authors.initials: J
authors.lastname: Quill
authors.forename: Paulo
authors.initials: P
authors.lastname: Vieira
authors.forename: Manuel
authors.initials: M
authors.lastname: Mota
authors.forename: Philippe
authors.initials: P
authors.lastname: Castagnone-Sereno