双重realtimePCR定量测定小麦条锈菌潜伏侵染方法的建立与应用

Establishment and application of duplex real-time PCR quantitative determination method on latent infection of wheat stripe rust

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, caused by Puccinia striiformis f. sp. tritici （PST）, is one of the most important wheat diseases in China. Based on two pairs of published primers （ from PST and wheat respectively）, we designed specific probes respectively and developed a duplex real-time PCR approach for the quantification of PST in latent period. In order to determine if the primers would interact in the duplex PCR system, and affect the system subsequently, we designed two real-time PCR reaction systems with a single pair of the primers and one probe from PST and wheat respectively for comparison. The linear regression analysis of CT values showed that duplex real-time PCR was hardly affected by the interaction of the primers. Serial ten-fold dilutions of samples to the determined concentration were performed for sensitivity test; the minimum genomic DNA concentrations of PST and wheat that could be accurately quantified were 0.4 pg and 0.5 ng respectively. In the mean time, two standard curves of PST and wheat were also set up. This method could be applied to detect samples from two different areas and to calculate molecular disease index （MDI）. Our results demonstrate that duplex real-time PCR is a reliable, more efficient and low error calculation method. It is therefore an optimized molecular method for detecting PST in latent period.