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Rapid preparation of Fusarium DNA from cereals for diagnostic PCR u sing sonification and an extraction kit
Author(s) -
Knoll S.,
Mulfinger S.,
Niessen L.,
Vogel R. F.
Publication year - 2002
Publication title -
plant pathology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.928
H-Index - 85
eISSN - 1365-3059
pISSN - 0032-0862
DOI - 10.1046/j.1365-3059.2002.00763.x
Subject(s) - fusarium , biology , trichothecene , dna extraction , lysis buffer , polymerase chain reaction , alkaline lysis , context (archaeology) , mycotoxin , variants of pcr , fusarium culmorum , microbiology and biotechnology , dna , botany , gene , genetics , plasmid , dna vaccination , paleontology
Several PCR methods have recently been developed for Fusarium analysis in pure cultures. To use these new techniques in mycological studies and in industrial quality control, a protocol was set up for the rapid preparation of fungal DNA from cereals. An ultrasonification probe (sonotrode) and a lysis buffer were used for mechanical lysis of mycelia from infected grains. Following ultrasonification, DNA was isolated using a commercially available kit. DNA preparation was completed within 5 min per sample. The method resulted in DNA of sufficient quality and quantity for diagnostic PCR. Group‐ and species‐specific primers were used to detect DNA of Fusarium graminearum and F. culmorum in species‐specific assays as well as trichothecene‐producing Fusarium spp. in a group‐specific system. A minimum of one F. graminearum ‐infected grain added to an uninfected 40 g wheat sample was detectable with a species‐specific PCR. The PCR signals produced with primers specific for the tri5 gene of trichothecene‐producing Fusarium spp. and with primers for the detection of F. graminearum (gaoA) were in accordance with corresponding concentrations of deoxynivalenol (DON) found in samples by HPLC analysis. The speed of the protocol developed may promote the use of PCR in routine applications in an agro‐industrial context.