Simultaneous detection of Fusarium culmorum and F. graminearum in plant material by duplex PCR with melting curve analysis.
BACKGROUND
Fusarium head blight (FHB) is a disease of cereal crops, which has a severe impact on wheat and barley production worldwide. Apart from reducing the yield and impairing grain quality, FHB leads to contamination of grain with toxic secondary metabolites (mycotoxins), which pose a health risk to humans and livestock. The Fusarium species primarily involved in FHB are F. graminearum and F. culmorum. A key prerequisite for a reduction in the incidence of FHB is an understanding of its epidemiology.


RESULTS
We describe a duplex-PCR-based method for the simultaneous detection of F. culmorum and F. graminearum in plant material. Species-specific PCR products are identified by melting curve analysis performed in a real-time thermocycler in the presence of the fluorescent dye SYBR Green I. In contrast to multiplex real-time PCR assays, the method does not use doubly labeled hybridization probes.


CONCLUSIONS
PCR with product differentiation by melting curve analysis offers a cost-effective means of qualitative analysis for the presence of F. culmorum and F. graminearum in plant material. This method is particularly suitable for epidemiological studies involving a large number of samples.
Brandfass
Christoph
C
University of Göttingen, Institute of Plant Pathology, Grisebachstrasse 6, 37077 Göttingen, Germany. cbrandf@gwdg.de
Karlovsky
Petr
P
D052160:Benzothiazoles; D017931:DNA Primers / Q000737:chemistry; D004271:DNA, Fungal / Q000737:chemistry / Q000302:isolation & purification; D003959:Diamines; D004587:Electrophoresis, Agar Gel / Q000379:methods; D005456:Fluorescent Dyes / Q000737:chemistry; D005506:Food Contamination / Q000032:analysis; D005516:Food Microbiology; D005670:Fusarium / Q000145:classification / Q000235:genetics / Q000302:isolation & purification*; D001467:Hordeum / Q000382:microbiology*; D009183:Mycotoxins / Q000032:analysis; D009930:Organic Chemicals / Q000737:chemistry; D010935:Plant Diseases / Q000382:microbiology; D016133:Polymerase Chain Reaction / Q000379:methods*; D011804:Quinolines; D012680:Sensitivity and Specificity; D013045:Species Specificity; D044366:Transition Temperature; D014908:Triticum / Q000382:microbiology*
D052160:Benzothiazoles; D017931:DNA Primers; D004271:DNA, Fungal; D003959:Diamines; D005456:Fluorescent Dyes; D009183:Mycotoxins; D009930:Organic Chemicals; D011804:Quinolines; C098022:SYBR Green I