基于TaqMan MGB探针的大豆北方茎溃疡病菌快速检测

Taq Man MGB-based Real-time Fluorescent PCR Method for The Rapid Detection of Diaporthe caulivora

为准确快速检测检疫性真菌大豆北方茎溃疡病菌,根据大豆北方茎溃疡病菌及其近似种的模式分离物ITS序列差异,设计并合成1对特异性引物和1条MGB探针,建立大豆北方茎溃疡病菌的实时荧光PCR检测方法。特异性试验结果表明:该检测方法能特异性检测大豆北方茎溃疡病菌。灵敏度试验结果表明:最低检测限量为10μL反应体系中总DNA含量1.0 pg;实时荧光PCR优化反应条件为引物终浓度0.5μmol·L^(-1),探针终浓度0.6μmol·L^(-1)。实际样品检测结果表明:该方法可用于疑似受大豆北方茎溃疡病菌侵染的进境大豆样品的检测与初筛。此方法准确、快速、灵敏,整个反应过程约1 h,检测过程完全闭管,无需PCR后续处理,可作为大豆北方茎溃疡病菌检测防控方法。

In order to accurately and rapidly detect the quarantine fungi Diaporthe caulivora,a species-specific real-time polymerase chain reaction(RT-PCR)assay was developed for the detection of D.caulivora.A pair of specific primers and a Taq Man-MGB probe were designed and synthesized according to the difference of ITS sequence between D.caulivora type and related species.A novel real-time fluorescent PCR was established to detect D.caulivora.The minimal detectable concentration of targeted DNA was 1.0 pg in 10μL reaction mixture.Optimal primer concentration and probe concentration were 0.5μmol·L^(-1)and 0.6μmol·L^(-1),respectively.The method could be used for the detection and preliminary screening of the samples suspected of carrying D.caulivora.The new method is more accurate,sensitive and time-saving than the traditional method and is suitable for routine use.It also provides a valuable tool for early rapid sensitive detection and identification of D.caulivora in plants and gives plant protection offices the chance to react accordingly and to evaluate measures for plant disease management against D.caulivora.