Validation and accreditation of a duplex real-time PCR test for reliable in planta detection of Chalara fraxinea1

This work reports the optimization and provides statistically supported validation data for a real‐time detection tool targeting the emerging fungus Chalara fraxinea . The test proved to be specific and inclusive, based on computer simulation and in vitro assessments, and could detect as little as 20 fg of C. fraxinea DNA. The duplex real‐time polymerase chain reaction assay proved to be significantly more efficient and rapid than isolation on agar plates. To monitor the performance of the whole analysis process and to ensure reliability of the results, a set of controls was used systematically during the assays. A diagnosis flow diagram is proposed. The Laboratoire National de la Protection des végétaux was recently accredited by the French accreditation body for this protocol.