RFLP在鳞翅目蛹虫草菌鉴别中的应用

Identification of Cordyceps militaris by Restriction Fragment Length Polymorphisms

本文应用RFLP技术鉴别两未知菌株S1001和S1002是否系鳞翅目蛹虫草菌(Cordyceps milicaris)。试验以ITSl,TTS4为引物,PCR扩增rDNA的5.8S+ITS区段,扩增产物纯化后用DNA限制性内切酶酶切,获得DNA的RFLP图谱,并同ATCC模式菌株的RFLP图谱相比较。结果表明:三个作为对照的ATC菌株具相同的RFLP图谱,而两待鉴定菌株之间存在差异,且明显不同于ATCC模式菌株,从而证明,两待鉴菌株与ATCC菌株之间在rDNA区域具有明显的结构差异,故推断两待鉴定菌株不隶属于鳞翅目蛹虫草菌。

RFLP was applied in order to identify if two unknown cultures--S1001,S1002were correctly assigned to Cordyceps militaris. Restriction analysis of enzymatically amplified ribosomal DNA was used to compare fingerprints of these cultures with three ATCC 'reference' strains of C. militaris. DNA was extracted from cultures grown in liquid medium and the region spanning of the 5.8S rRNA gene and the two internal transcribed spaces (5.8S + ITS) were amplified with the Polymerase Chain Reaction (PCR). The PCR products were purified and then subjected to double digestion with three pairs of cndonucleases. For all enzyme pairs, the three ATCC strains exhibited identical profiles that were easily distinguishable from those of the two unknown strains, which showed very different fingerprints. The results suggested that the two cult-arts were incorrectly assigned to C. militaris.