Leisova L, Minarikova V, Kucera L, Ovesna J
Research Institute of Crop Production, Drnovska 507, 16106 Prague 6 Ruzyne, Czech Republic.
J Microbiol Methods. 2006 Dec;67(3):446-55. doi: 10.1016/j.mimet.2006.04.018. Epub 2006 Jun 30.
Net blotch is a barley foliar disease caused by two forms of Pyrenophora teres: Pyrenophora teres f. teres (PTT) and Pyrenophora teres f. maculata (PTM). To monitor and quantify their occurrence during the growing season, diagnostic system based on real-time PCR was developed. TaqMan MGB (Minor Groove Binder) primers and probes were designed that showed high specificity for each of the two forms of P. teres. As a host plant internal standard, TaqMan MGB primers and probe based on RacB gene sequence were designed. The method was optimised on pure fungal DNA and on plasmid standard dilutions. Quantification was accomplished by comparing Ct values of unknown samples with those obtained from plasmid standard dilutions. The assay detects down to five gene copies per reaction. It is able to produce reliable quantitative data over a range of six orders of magnitude. The developed assay was used to differentiate and quantify both forms of P. teres in infected barley leaves. Correlation R(2)=0.52 was obtained between the Ct values and size of symptoms areas in early stage of infection. Application of the TaqMan MGB technology to leaf samples collected in 20 barley varieties in the region Kromeriz during the growing season of 2003 and 2004 revealed that P. teres f. teres predominated in these 2 years. The developed method is an important tool to quantify and monitor the dynamics of the two forms of P. teres during the growing season.
圆核腔菌圆核腔专化型（PTT）和圆核腔菌黄斑专化型（PTM）。为了监测和量化它们在生长季节的发生情况，开发了基于实时PCR的诊断系统。设计了TaqMan MGB（小沟结合剂）引物和探针，它们对两种形式的圆核腔菌均表现出高特异性。作为宿主植物内标，设计了基于RacB基因序列的TaqMan MGB引物和探针。该方法在纯真菌DNA和质粒标准稀释液上进行了优化。通过将未知样品的Ct值与从质粒标准稀释液获得的Ct值进行比较来完成定量。该检测方法每个反应能检测低至5个基因拷贝。它能够在六个数量级的范围内产生可靠的定量数据。所开发的检测方法用于区分和定量感染大麦叶片中两种形式的圆核腔菌。在感染早期，Ct值与症状面积大小之间的相关系数R² = 0.52。在2003年和2004年生长季节，将TaqMan MGB技术应用于在克罗梅日什地区收集的20个大麦品种的叶片样品，结果表明在这两年中圆核腔菌圆核腔专化型占主导。所开发的方法是在生长季节量化和监测两种形式的圆核腔菌动态的重要工具。
