Open AccessSatellite DNA as a target for TaqMan real‐time PCR detection of the pinewood nematode, Bursaphelenchus xylophilus
Author(s) -
FRANÇOIS CECILE,
CASTAG CHANTAL,
BOONHAM NEIL,
TOMLINSON JENNY,
LAWSON REBECCA,
HOCKLAND SUE,
QUILL JAMES,
VIEIRA PAULO,
MOTA MANUEL,
CASTAGSERENO PHILIPPE
Publication year - 2007
Publication title -
molecular plant pathology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.945
H-Index - 103
eISSN - 1364-3703
pISSN - 1464-6722
DOI - 10.1111/j.1364-3703.2007.00434.x
Subject(s) - bursaphelenchus xylophilus , biology , taqman , dna extraction , satellite dna , xylophilus , genomic dna , nematode , polymerase chain reaction , real time polymerase chain reaction , dna , genome , computational biology , genetics , gene , ecology
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 Msp I 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.