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A duplex PCR method for identification of cultures of Fusarium graminearum from infected wheat grain without DNA extraction
- Zuzak, Krista, Zahr, Kher, Yang, Yalong, Sarkes, Alian, Feindel, David, Daniels, Greg, Harding, Michael W., Feng, Jie
- Canadian journal of plant pathology 2018 v.40 no.3 pp. 417-422
- DNA, Fusarium graminearum, denaturation, nucleic acid annealing, oligodeoxyribonucleotides, polymerase chain reaction, protocols, quality control, wheat
- A simple and accurate PCR protocol for identification of Fusarium graminearum from fungal cultures was developed. In this protocol, DNA extraction was omitted by using the Thermo Scientific Phire direct PCR master mix kit for template preparation and PCR. Two primer pairs, ITS5/ITS2 for DNA template quality control and Fg16F/Fg16R for F. graminearum identification, were used in a duplex PCR. Each 20-µL PCR contained 10 µL 2× Phire Direct PCR master mix, 2 µL template and 0.25 µM each of the four primers. The PCR program consisted of an initial denaturation at 98°C for 5 min, followed by 40 cycles of denaturation at 98°C for 5 s, annealing at 62°C for 5 s and extension at 72°C for 20 s, and a final extension at 72°C for 1 min. This protocol was used to test 196 fungal cultures isolated from 75 wheat samples. Fourteen cultures were identified as F. graminearum. This protocol was demonstrated to be simpler and more accurate than other PCR protocols for F. graminearum identification.