Kianianmomeni Arash, Schwarz Gerhard, Felsenstein Friedrich G, Wenzel Gerhard
Department of Plant Science, Centre of Life and Food Sciences Weihenstephan, Technical University Munich, D-85350 Freising, Germany.
Pest Manag Sci. 2007 Mar;63(3):219-24. doi: 10.1002/ps.1290.
A single nucleotide polymorphism (SNP) in the cytochrome b gene confers resistance to strobilurin fungicides for several fungal pathogens. Therefore, on the basis of a change at amino acid position 143 from glycine to alanine, a real-time PCR assay was established for the quantitative detection of the analogous SNP in the cytochrome b sequence of Pyrenophora teres Drechsler, which causes barley net blotch. Allelic discrimination was achieved by using allele specific primers with artificially mismatched nucleic acid bases and minor groove binding probes. Validation parameters for the lower limits of the working range, namely limits of detection (LOD) and limits of quantification (LOQ), were statistically determined by the variance of calibration data, as well as by the variance of the 100% non-strobilurin-resistant allele DNA sample (blank values). It was found that the detection was limited by the variance of blank values (five in 801 458 copies; 0.0006%), whereas the quantification was limited by the variance of calibration data (37 in 801 458 copies; 0.0046%). The real-time PCR assay was finally used to monitor strobilurin-resistant cytochrome b alleles in barley net blotch field samples, which were already classified in in vivo biotests to be fully sensitive to strobilurins. All signals for strobilurin-resistant cytochrome b alleles were below the LOD, and therefore the results are in total agreement with the phenotypes revealed by biotests.
细胞色素b基因中的单核苷酸多态性（SNP）赋予了几种真菌病原体对甲氧基丙烯酸酯类杀菌剂的抗性。因此，基于氨基酸位置143处从甘氨酸到丙氨酸的变化，建立了一种实时荧光定量PCR检测方法，用于定量检测引起大麦网斑病的圆核腔菌（Pyrenophora teres Drechsler）细胞色素b序列中的类似SNP。通过使用具有人工错配核酸碱基的等位基因特异性引物和小沟结合探针实现等位基因鉴别。工作范围下限的验证参数，即检测限（LOD）和定量限（LOQ），通过校准数据的方差以及100%对甲氧基丙烯酸酯类杀菌剂不耐药的等位基因DNA样本（空白值）的方差进行统计学确定。发现检测受空白值方差的限制（801458个拷贝中有5个；0.0006%），而定量受校准数据方差的限制（801458个拷贝中有37个；0.0046%）。最终，该实时荧光定量PCR检测方法用于监测大麦网斑病田间样本中对甲氧基丙烯酸酯类杀菌剂耐药的细胞色素b等位基因，这些样本在体内生物测试中已被分类为对甲氧基丙烯酸酯类杀菌剂完全敏感。所有对甲氧基丙烯酸酯类杀菌剂耐药的细胞色素b等位基因的信号均低于检测限，因此结果与生物测试揭示的表型完全一致。
