Cryphonectria parasitica is a fungal pathogen that causes lethal bark necrosis in chestnut. A duplex qPCR allowing detection of the pathogen and its host, Castanea sativa, is described. The method can be used for early detection of the pathogen in chestnut bark tissues with an internal control of false-negative results caused by PCR inhibitors and/or DNA extraction failure. A positive amplification control of qPCR that allows detection of any deviation from a normal qPCR run based on a control chart is also described. As C. parasitica is a regulated pathogen in Europe, the protocol also provides information on the way to collect and handle bark samples to fulfil biosecurity rules.
Keywords: Chestnut blight; Endophyte; Fungal pathogen; Internal amplification control; Positive amplification control; Real-time PCR.
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