Phytopathology. 1999 Sep;89(9):796-804. doi: 10.1094/PHYTO.1999.89.9.796.
ABSTRACT Species-specific detection of Diaporthe phaseolorum and Phomopsis longicolla from soybean seeds was accomplished using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and TaqMan chemistry. To use these detection systems, fungal DNA was released from soybean seed coats using an ultrasonic processor to break the cells. DNA fragment lengths ranged from 200 to 1,200 base pairs (bp), with the majority of fragments <500 bp. Based on DNA sequences of the internal transcribed spacer (ITS) regions of ribosomal DNA, three TaqMan primer/probe sets were designed. Primer/probe set PL-5 amplified a 96-bp fragment within the ITS1 region of P. longicolla, D. phaseolorum var. caulivora, D. phaseolorum var. meridionalis, and D. phaseolorum var. sojae. Set PL-3 amplified a 86-bp DNA fragment within the ITS2 region of P. longicolla. Set DPC-3 amplified a 151-bp DNA fragment within the ITS2 region of D. phaseolorum var. caulivora. TaqMan primer/probe sets were able to detect as little as 0.15 fg (four copies) of plasmid DNA. When using PCR-RFLP for Diaporthe and Phomopsis detection, the sensitivity was as low as 100 pg of pure DNA. Among 13 soybean seed lots from Italy and the United States, the total Diaporthe and Phomopsis detected using a traditional seed-plating technique ranged from 0 to 32%. P. longicolla was most prevalent, followed by D. phaseolorum var. sojae. D. phaseolorum var. caulivora, which only occurred in 0.5% of the Italian seed lots, was not detected in the U.S. seed lots. D. phaseolorum var. meridionalis was not detected in either the U.S. or Italian seed lots. Using TaqMan primer/probe set PL-3, the frequency of P. longicolla was 18% in seed lot I3, similar to the frequency obtained from PCR-RFLP and potato dextrose agar plating detection. The frequencies of D. phaseolorum and P. longicolla in each seed lot obtained by the different detection methods were comparable with respect to total infection and individual species detection. However, TaqMan detection provided the fastest results of all the methods tested.
摘要 采用聚合酶链反应-限制性片段长度多态性（PCR-RFLP）和 TaqMan 化学方法，从大豆种子中特异性检测到 Diaporthe phaseolorum 和 Phomopsis longicolla。为了使用这些检测系统，使用超声波处理器从大豆种皮中释放真菌 DNA，以打破细胞。DNA 片段长度范围为 200 至 1200 个碱基对（bp），大多数片段<500 bp。基于核糖体 DNA 的内部转录间隔区（ITS）序列的 DNA 序列，设计了三个 TaqMan 引物/探针组。引物/探针组 PL-5 扩增了 P. longicolla、D. phaseolorum var. caulivora、D. phaseolorum var. meridionalis 和 D. phaseolorum var. sojae 的 ITS1 区的 96-bp 片段。PL-3 组扩增了 P. longicolla 的 ITS2 区的 86-bp DNA 片段。DPC-3 组扩增了 D. phaseolorum var. caulivora 的 ITS2 区的 151-bp DNA 片段。TaqMan 引物/探针组能够检测到低至 0.15 fg（四个拷贝）的质粒 DNA。使用 PCR-RFLP 检测 Diaporthe 和 Phomopsis 时，灵敏度低至 100 pg 纯 DNA。在来自意大利和美国的 13 个大豆种子批中，使用传统种子平板技术检测到的总 Diaporthe 和 Phomopsis 范围为 0 至 32%。P. longicolla 最为普遍，其次是 D. phaseolorum var. sojae。仅在 0.5%的意大利种子批中发现的 D. phaseolorum var. caulivora 未在美国种子批中检测到。D. phaseolorum var. meridionalis 未在美国或意大利种子批中检测到。使用 TaqMan 引物/探针组 PL-3，种子批 I3 中 P. longicolla 的频率为 18%，与从 PCR-RFLP 和土豆葡萄糖琼脂平板检测获得的频率相似。不同检测方法获得的每个种子批中 D. phaseolorum 和 P. longicolla 的频率在总感染和单个物种检测方面与总感染和单个物种检测相当。然而，与所有测试方法相比，TaqMan 检测提供了最快的结果。
