Development of a highly sensitive nested-PCR method using a single closed tube for detection of Fusarium culmorum in cereal samples

Aims: The aim of the study was to develop a sensitive detection method of Fusarium culmorum contamination in cereal samples. Methods and Results: A nested-PCR method using a single closed tube was developed for the detection of F. culmorum in infected cereal samples. The concentrations of the first primer pair was diluted 10 000 times compared to the concentration used for the second primer pair. Differing annealing temperatures allowed both first and second polymerase chain reaction (PCR) reactions to be performed subsequently in the same closed tube. The detection limit was 5-50 fg of purified target DNA and allowed the detection of 1% infected seeds of wheat in a mixture with uninfected grains. Conclusions: F. culmorum can be specifically detected in cereal samples by the highly sensitive method of nested-PCR in a single closed tube. Significance and Impact of the Study: This work describes the detection of F. culmorum in cereal samples that is approximately 100 times more sensitive than previous PCR methods, involves low risk of cross contaminations between samples, low costs and reduced hands-on time as compared to standard nested-PCR protocols.