Rollo F, Salvi R, Torchia P
Dipartimento di Biologia Cellulare, Camerino, Italy.
Appl Microbiol Biotechnol. 1990 Feb;32(5):572-6. doi: 10.1007/BF00173730.
A new method for the diagnosis of the plant pathogenic fungus Phoma tracheiphila has been developed. The method takes advantage of the enzymatic amplification of a specific 102 bp-long target sequence of fungal DNA by the polymerase chain reaction (PCR) using Thermus aquaticus DNA polymerase. The amplified DNA was characterized by agarose-gel electrophoresis, molecular hybridization using a synthetic oligonucleotide probe and direct sequencing. The application of the new method makes possible fast and direct detection of the pathogen in lignified plant tissues, a goal not previously achieved when a cloned probe and a dot-blot test were employed. In addition the PCR test can be used to advantage as a particularly simple and fast way of typing fungal isolates. This is achieved by submitting to DNA amplification crude homogenates of fungal mycelium and analysing the amplified DNA on an agarose mini-gel.
已开发出一种诊断植物致病真菌葡萄座腔菌的新方法。该方法利用水生栖热菌DNA聚合酶，通过聚合酶链反应（PCR）对真菌DNA特定的102 bp长靶序列进行酶促扩增。扩增的DNA通过琼脂糖凝胶电泳、使用合成寡核苷酸探针的分子杂交和直接测序进行表征。新方法的应用使得在木质化植物组织中快速直接检测病原体成为可能，这是使用克隆探针和斑点印迹试验时以前无法实现的目标。此外，PCR试验可作为一种特别简单快速的真菌分离株分型方法加以利用。这是通过将真菌菌丝体的粗匀浆进行DNA扩增，并在琼脂糖微型凝胶上分析扩增的DNA来实现的。
