Deora Abhinandan, Gossen Bruce D, Amirsadeghi Sasan, McDonald Mary Ruth
Department of Plant Agriculture, University of Guelph, ON N1G 2W1, Canada.
Agriculture and Agri-Food Canada, Saskatoon Research Centre, SK S7N 0X2, Canada.
Plant Dis. 2015 Jul;99(7):1002-1009. doi: 10.1094/PDIS-06-14-0608-RE. Epub 2015 May 22.
Various physical and chemical factors in soil can inhibit the detection and quantification of soilborne plant pathogens using quantitative polymerase chain reaction (qPCR) assays. A multiplexed TaqMan qPCR assay, including a competitive internal positive control (CIPC), was developed to identify and (where necessary) compensate for inhibition in the quantification of resting spores of Plasmodiophora brassicae from soil. The CIPC amplicon was developed by modifying a sequence coding for green fluorescent protein so that it could be amplified with P. brassicae-specific primers. Addition of CIPC at 5 fg/μl to the singleplex qPCR assay designed to quantify P. brassicae genomic DNA did not reduce the sensitivity, specificity, or reproducibility of the assay. Each of the soil samples, either artificially inoculated or naturally infested with P. brassicae, exhibited no amplification of the CIPC. When the samples were diluted and reassessed, the quantification cycle of the CIPC relative to the control (water only) was delayed in each sample. The magnitude of the delay was used to adjust the estimate of resting spore concentration. The corrected concentration estimates were significantly higher than the unadjusted estimate, which indicated the presence of DNA inhibitors in samples even after dilution. The only exception was a mineral soil sample inoculated with a low concentration (10 spores/g) of resting spores. The assay was optimized for use on a range of soil types. A sample of 0.25 g for mineral soil and 0.10 g for high-organic-matter soil was optimum for recovery of DNA of P. brassicae. The assay represents an improvement over existing assays for estimating resting spore concentration in infested fields.
土壤中的各种物理和化学因素会抑制利用定量聚合酶链反应（qPCR）测定法对土壤传播的植物病原体进行检测和定量。开发了一种多重TaqMan qPCR测定法，包括竞争性内部阳性对照（CIPC），用于鉴定并在必要时补偿从土壤中定量芸薹根肿菌休眠孢子时的抑制作用。通过修饰编码绿色荧光蛋白的序列来开发CIPC扩增子，以便它可以用芸薹根肿菌特异性引物进行扩增。在旨在定量芸薹根肿菌基因组DNA的单重qPCR测定法中添加5 fg/μl的CIPC不会降低该测定法的灵敏度、特异性或可重复性。每个要么人工接种要么自然感染芸薹根肿菌的土壤样品均未显示出CIPC的扩增。当对样品进行稀释并重新评估时，每个样品中CIPC相对于对照（仅水）的定量循环均延迟。延迟的幅度用于调整休眠孢子浓度的估计值。校正后的浓度估计值明显高于未调整的估计值，这表明即使在稀释后样品中仍存在DNA抑制剂。唯一的例外是接种了低浓度（10个孢子/克）休眠孢子的矿质土壤样品。该测定法针对一系列土壤类型进行了优化。对于矿质土壤，0.25克的样品以及对于高有机质土壤，0.10克的样品最适合回收芸薹根肿菌的DNA。该测定法相对于现有测定法在估计受侵染田地中休眠孢子浓度方面有所改进。
