title: Method evaluation of Fusarium DNA extraction from mycelia and wheat for down-stream real-time PCR quantification and correlation to mycotoxin levels.
abstract: Identification of Fusarium species by traditional methods requires specific skill and experience and there is an increased interest for new molecular methods for identification and quantification of Fusarium from food and feed samples. Real-time PCR with probe technology (Taqman) can be used for the identification and quantification of several species of Fusarium from cereal grain samples. There are several critical steps that need to be considered when establishing a real-time PCR-based method for DNA quantification, including extraction of DNA from the samples. In this study, several DNA extraction methods were evaluated, including the DNeasy Plant Mini Spin Columns (Qiagen), the Bio robot EZ1 (Qiagen) with the DNeasy Blood and Tissue Kit (Qiagen), and the Fast-DNA Spin Kit for Soil (Qbiogene). Parameters such as DNA quality and stability, PCR inhibitors, and PCR efficiency were investigated. Our results showed that all methods gave good PCR efficiency (above 90%) and DNA stability whereas the DNeasy Plant Mini Spin Columns in combination with sonication gave the best results with respect to Fusarium DNA yield. The modified DNeasy Plant Mini Spin protocol was used to analyse 31 wheat samples for the presence of F. graminearum and F. culmorum. The DNA level of F. graminearum could be correlated to the level of DON (r(2) = 0.9) and ZEN (r(2) = 0.6) whereas no correlation was found between F. culmorum and DON/ZEA. This shows that F. graminearum and not F. culmorum, was the main producer of DON in Swedish wheat during 2006.
authors.affiliation: National Food Administration, P.O Box 622, SE-751 26 Uppsala, Sweden. Elisabeth.Fredlund@slv.es
authors.forename: Elisabeth
authors.initials: E
authors.lastname: Fredlund
authors.forename: Ann
authors.initials: A
authors.lastname: Gidlund
authors.forename: Monica
authors.initials: M
authors.lastname: Olsen
authors.forename: Thomas
authors.initials: T
authors.lastname: Börjesson
authors.forename: Niels Henrik Hytte
authors.initials: NH
authors.lastname: Spliid
authors.forename: Magnus
authors.initials: M
authors.lastname: Simonsson
mesh_terms: D004271:DNA, Fungal / Q000032:analysis / Q000235:genetics / Q000302:isolation & purification*; D005516:Food Microbiology; D005670:Fusarium / Q000737:chemistry / Q000145:classification / Q000235:genetics / Q000302:isolation & purification*; D025282:Mycelium / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D009172:Mycology / Q000379:methods*; D009183:Mycotoxins / Q000032:analysis* / Q000378:metabolism; D016133:Polymerase Chain Reaction; D014255:Trichothecenes / Q000032:analysis / Q000378:metabolism; D014908:Triticum / Q000382:microbiology*; D015025:Zearalenone / Q000032:analysis / Q000378:metabolism
chemical_list: D004271:DNA, Fungal; D009183:Mycotoxins; D014255:Trichothecenes; D015025:Zearalenone; C007262:deoxynivalenol