Phytopathology. 2000 Oct;90(10):1137-44. doi: 10.1094/PHYTO.2000.90.10.1137.
ABSTRACT Polymerase chain reaction (PCR) products were identified and amplified from isolates of Aphanomyces euteiches and A. cochlioides. The products were cloned and sequenced, and the data were used to design pairs of extended PCR primers to amplify sequence-characterized DNA markers. The primer pair OPC7-FS-30 and OPC7-RS-25 amplified a single 1,332-bp product from all isolates of A. euteiches that were not amplified from any other isolates tested. A single 718-bp product was selectively amplified only from isolates of A. cochlioides with the primer pair OPB10-FS-25 and OPB10-RS-25. A. euteiches was detected in roots of several varieties of field-grown peas collected from a root rot trial site. PCR also detected A. euteiches in the organic fraction of field soil samples. Both pairs of extended primers were used in a multiplex reaction to unambiguously discriminate between A. euteiches and A. cochlioides. Both pairs of primers were used in two-step PCR reactions in which annealing and extension was done in a single step at 72 degrees C. This reduced the time required for amplification of the diagnostic PCR product and its resolution by electrophoresis to less than 3 h.
摘要 从串珠镰刀菌（Aphanomyces euteiches）和禾旋孢腔菌（A. cochlioides）的分离物中鉴定和扩增了聚合酶链反应（PCR）产物。对产物进行了克隆和测序，并利用这些数据设计了两对扩展 PCR 引物来扩增具有序列特征的 DNA 标记。引物对 OPC7-FS-30 和 OPC7-RS-25 从所有未被任何其他测试分离物扩增的 A. euteiches 分离物中扩增出单一的 1332bp 产物。引物对 OPB10-FS-25 和 OPB10-RS-25 仅从禾旋孢腔菌的分离物中选择性扩增出单一的 718bp 产物。在根腐病试验现场采集的几种田间豌豆品种的根系中检测到了串珠镰刀菌。PCR 还在田间土壤样本的有机部分检测到了串珠镰刀菌。两对扩展引物都用于多重反应中，可明确区分串珠镰刀菌和禾旋孢腔菌。这两对引物都用于两步 PCR 反应中，其中退火和延伸在 72℃下一步完成。这将扩增诊断性 PCR 产物及其电泳分辨率所需的时间减少到 3 小时以内。
