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小麦条锈病是我国小麦生产上的重要病害之一。为高效准确地定量检测处于潜伏侵染阶段的小麦条锈菌,本研究根据已发表的小麦条锈菌和寄主小麦的引物,设计了各自的探针,建立了双重real-time PCR检测方法。为排除多个引物互作造成的干扰,对小麦条锈菌引物探针体系、小麦体系以及二者的双重real-time PCR体系进行了比较。CT值相关线性回归分析证明,引物之间互作很小,对定量检测无影响。已知浓度样品经梯度稀释,进行灵敏度检测,确定了双重real-time PCR对小麦条锈菌DNA和小麦DNA准确定量测定的最小检测限为0.4 pg和0.5 ng。同时建立了小麦条锈菌和小麦各自的标准曲线。用此方法检测来自两个不同地区的田间样本,得到的分子病情指数(MDI)与随后的发病趋势一致。本研究建立的双重real-time PCR分析方法可靠、高效、低误差,是对本实验室已有小麦条锈菌潜伏期分子检测方法的进一步优化。
Wheat stripe rust is one of the most important diseases in wheat production in our country. In order to efficiently and accurately detect wheat stripe rust in the stage of latent infection, two probes were designed according to the published primers of wheat stripe rust and host wheat, and a dual real-time PCR method was established. In order to eliminate the interference caused by the interaction of multiple primers, the double real-time PCR system of wheat stripe rust primer probe system, wheat system and the two were compared. Linear regression analysis of CT values showed that the interaction between primers was small and had no effect on quantitative detection. The known concentration of the sample was subjected to gradient dilution for sensitivity detection. The minimum detection limits of double real-time PCR for the accurate quantitative determination of DNA and wheat DNA were determined to be 0.4 pg and 0.5 ng, respectively. At the same time, the respective standard curves of wheat stripe rust and wheat were established. Using this method to detect field samples from two different regions, the molecular disease index (MDI) obtained was consistent with the subsequent trend of onset. The double real-time PCR assay established in this study is reliable, efficient and low-error. It is a further optimization of the molecular detection method of latency of P. stripelii in our laboratory.