Detection of Fusarium culmorum in wheat by a surface plasmon resonance-based DNA sensor.
A surface plasmon resonance (SPR) sensor based on DNA hybridization has been developed for the detection of Fusarium culmorum, a fungal pathogen of cereals. A 0.57 kbp DNA fragment of F. culmorum was amplified by specific primers and a 25-mer oligonucleotide probe was selected within the sequence of the PCR amplicon. After biotinilation, the probe was immobilized on a streptavidin sensor chip and tested for biospecific interaction with PCR products of F. culmorum. The effect of denaturating agents (formamide and urea) and ionic strength (NaCl) on hybridization efficiency of double-stranded PCR products with the immobilized probe and the specificity of the probe were investigated. The SPR biosensor was successfully used for the detection of F. culmorum in culture material of different strains and in naturally infected wheat samples. Tested on fungal cultures, it showed a good selectivity for F. culmorum against other species of either Fusarium or other fungal genera. A background signal was observed in wheat samples strictly depending on the DNA amount of the testing matrix. Testing 30 ng of durum wheat DNA the detection limit was 0.06 pg of F. culmorum DNA. The developed PCR-SPR assay allowed to detect F. culmorum with sensitivity and specificity higher than gel-electrophoresis analysis.
Zezza
Francesco
F
Institute of Sciences of Food Production (ISPA), National Research Council, Via G. Amendola 122/0, 70126 Bari, Italy. francesco.zezza@ispa.cnr.it
Pascale
Michelangelo
M
Mulè
Giuseppina
G
Visconti
Angelo
A
D015374:Biosensing Techniques / Q000379:methods; D004271:DNA, Fungal / Q000737:chemistry / Q000235:genetics; D005670:Fusarium / Q000235:genetics* / Q000302:isolation & purification; D010935:Plant Diseases / Q000382:microbiology*; D016133:Polymerase Chain Reaction; D020349:Surface Plasmon Resonance / Q000379:methods*; D014908:Triticum
D004271:DNA, Fungal