Niepold F, Schöber-Butin B
Biologische Bundesanstalt für Land- und Forstwirtschaft, Institut für Pflanzenschutz in Ackerbau und Grünland, Braunschweig.
Microbiol Res. 1995 Nov;150(4):379-85. doi: 10.1016/S0944-5013(11)80020-0.
A characterized repetitive sequence from Phytophthora infestans (P. infestans) was used to perform a PCR with the DNA from the four races 1, 3, 4, and 1-11. To obtain amplifiable DNA, all extractions had to be purified by DNA adsorbing spin columns. Only two out of four tested primers were well suited and gave an DNA amplificate of the same size for all four races. After optimization the detection limit of the PCR corresponded to 100 ng of freeze-dried mycelia per ml, specificity was established when testing a collection of the most important potato pathogenic fungi and bacteria. Using P. infestans zoospores to infect tuber slices, the detection threshold was determined to be two days post infection when 3 to 6 zoospores were applied. After infiltrating the tenfold concentration into potato leaves a visible PCR signal was obtained one day post infection. Further improvements of the sensitivity threshold in detecting P. infestans for breeding and prognosis purposes are discussed.
利用来自致病疫霉（P. infestans）的一个特征性重复序列，对1、3、4和1-11这四个生理小种的DNA进行PCR扩增。为了获得可扩增的DNA，所有提取物都必须通过DNA吸附旋转柱进行纯化。在四个测试引物中，只有两个非常合适，并且对所有四个生理小种都产生了相同大小的DNA扩增产物。优化后，PCR的检测限相当于每毫升100 ng冻干菌丝体，在检测一组最重要的马铃薯致病真菌和细菌时确定了其特异性。使用致病疫霉游动孢子感染薯块切片，当接种3至6个游动孢子时，检测阈值确定为感染后两天。将十倍浓度的菌液渗入马铃薯叶片后，感染后一天获得了可见的PCR信号。文中还讨论了为育种和预后目的在检测致病疫霉方面进一步提高灵敏度阈值的问题。
