The rapid identification of European Armillaria species from soil samples by nested PCR.
New specific primers AR1 and AR2 were successfully used for the amplification of a specific part of internal transcribed spacer (ITS) of rDNA of Armillaria isolated from soil samples. DNA was isolated from 0.5 g of forest soil and ITS region was amplified by nested PCR reaction with external primers ITS1 and ITS4 and internal primers AR1 and AR2. The individual species were distinguished by restriction fragment length polymorphisms (RFLPs) analysis with restriction endonuclease HinfI. The fragments were analysed by ion-exchange HPLC that is more sensible and more rapid than electrophoresis. The amplicons were sequenced to improve the discrimination between the species. The method enables the identification of Armillaria species within one day directly from soil samples without the need for previous isolation and cultivation of mycelium of Armillaria.
Department of Biochemistry, Faculty of Science, Masaryk University, Kotlarska 2, 61137 Brno, Czech Republic.
Jan
J
Lochman
Omar
O
Sery
Vladimir
V
Mikes
D000363:Agaricales / Q000145:classification* / Q000235:genetics / Q000302:isolation & purification*; D002851:Chromatography, High Pressure Liquid; D004271:DNA, Fungal / Q000032:analysis / Q000302:isolation & purification / Q000378:metabolism; D021903:DNA, Ribosomal Spacer / Q000032:analysis / Q000302:isolation & purification / Q000378:metabolism; D015252:Deoxyribonucleases, Type II Site-Specific / Q000378:metabolism; D016133:Polymerase Chain Reaction; D012150:Polymorphism, Restriction Fragment Length; D012680:Sensitivity and Specificity; D017422:Sequence Analysis, DNA; D012988:Soil Microbiology
D004271:DNA, Fungal; D021903:DNA, Ribosomal Spacer; D015252:Deoxyribonucleases, Type II Site-Specific; C032229:GANTC-specific type II deoxyribonucleases