Rapid and accurate detection of Ceratocystis fagacearum from stained wood and soil by nested and real-time PCR
A nested and real-time PCR assay was developed for the rapid and accurate detection of Ceratocystis fagacearum, which is the causal agent of oak wilt in stained wood and soil. Based on the differences of the internal transcribed spacer (ITS) sequences of Ceratocystis spp., one pair of species-specific primers, CF01/CF02, was designed. Whereas a 280-bp product was amplified using the purified DNA from three isolates of C. fagacearum as the template, no PCR product was obtained from template of other 18 fungi. The detection sensitivity was 10 pg genomic DNA per 25-μl PCR reaction volume. To increase detection sensitivity, a nested PCR was developed by using ITS1/ITS4 as the first-round primers and CF01/CF02 in the second round, as it can detect 1 pg genomic DNA per 25-μl PCR reaction volume. More importantly, CF01/CF02 primers were successfully adapted to real-time PCR with a detection limit of 0.1 pg genomic DNA per 20-μl PCR reaction volume. Using these two methods, we could rapidly and accurately detect the pathogen in artificially infected wood and soil.
Document Type: Research Article
Affiliations: 1: Jiangsu Province Key Laboratory for Prevention and Management of Invasive Species, Nanjing Forest University, Nanjing 210037, China 2: Plant Quarantine Laboratory, Jiangsu Entry-Exit Inspection and Quarantine Bureau, Nanjing, China
Publication date: 01 February 2011