Lochman Jan, Sery Omar, Mikes Vladimir
Department of Biochemistry, Faculty of Science, Masaryk University, Kotlarska 2, 61137 Brno, Czech Republic.
FEMS Microbiol Lett. 2004 Aug 1;237(1):105-10. doi: 10.1016/j.femsle.2004.06.019.
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.
新的特异性引物AR1和AR2成功用于扩增从土壤样品中分离出的蜜环菌核糖体DNA内转录间隔区（ITS）的特定部分。从0.5克森林土壤中提取DNA，通过巢式PCR反应，使用外部引物ITS1和ITS4以及内部引物AR1和AR2扩增ITS区域。通过用限制性内切酶HinfI进行限制性片段长度多态性（RFLP）分析来区分各个物种。通过离子交换高效液相色谱法分析片段，该方法比电泳更灵敏、更快速。对扩增子进行测序以提高物种间的鉴别能力。该方法能够在一天内直接从土壤样品中鉴定出蜜环菌物种，而无需事先分离和培养蜜环菌的菌丝体。
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