Phytopathology. 2001 Sep;91(9):900-4. doi: 10.1094/PHYTO.2001.91.9.900.
ABSTRACT A technique based on the polymerase chain reaction (PCR) was developed for the identification of Venturia nashicola using nucleotide sequence information of the ribosomal DNA region. The complete internal transcribed spacer (ITS) region of V. nashicola strains and phylo-genetically related species was amplified with the two universal ITS1 and ITS4 primers, sequenced, and digested with five restriction enzymes. The alignment of nucleotide sequences and analyses of digestion patterns indicated constant polymorphisms between V. nashicola and related species at nucleotides 126 and 127, which overlapped a TaqI restriction site. An oligonucleotide primer named A126 was designed for identifying this variable region. A primer set (A126 and ITS4) that allowed the amplification of a 391-bp DNA fragment within the ITS region by PCR was specific to V. nashicola when it was checked against fungal genomic DNAs of related fungi. This primer set was a good candidate for a species-specific reagent in a procedure for identification of V. nashicola by PCR.
摘要 本研究利用核糖体 DNA 区的核苷酸序列信息，开发了一种基于聚合酶链反应（PCR）的鉴定梨黑星病菌的技术。使用通用 ITS1 和 ITS4 引物扩增梨黑星病菌株和系统发育相关物种的完整内部转录间隔区（ITS），对扩增产物进行测序，并使用 5 种限制酶进行消化。核苷酸序列比对和消化图谱分析表明，在核苷酸 126 和 127 处，梨黑星病菌与相关物种之间存在恒定的多态性，该区域重叠 TaqI 限制位点。设计了一个名为 A126 的寡核苷酸引物来鉴定这个可变区域。当用相关真菌的真菌基因组 DNA 进行检查时，引物对 A126 和 ITS4 可通过 PCR 扩增 ITS 区的 391bp DNA 片段，对梨黑星病菌具有特异性。该引物对是 PCR 鉴定梨黑星病菌的种特异性试剂的良好候选物。
