烟草靶斑病菌LFD-RAA快速检测方法的建立
A rapid LFD-RAA detection method for tobacco target spot pathogen
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摘要:
为快速有效检测烟草靶斑病菌,以烟草靶斑病菌beta-tubulin基因序列保守区域为靶标位点,设计重组酶介导扩增技术(Recombinase aided amplification,RAA)引物,结合侧流层析试纸条(Lateral flow dipstick,LFD),建立烟草靶斑病菌快速检测体系并进行优化。结果表明,LFD-RAA检测体系在39 ℃恒温条件下反应30 min即可完成RAA扩增,并具有较高的特异性,其检测极限值为100 fg/μL,与PCR检测灵敏度相当。对检测体系优化确定检测扩增的最优时间为20~35 min、扩增温度为31~39 ℃。LFD-RAA检测体系可应用于烟草上烟草靶斑病菌的快速检测。
Abstract:To rapidly and effectively detect the tobacco target spot pathogen, the conserved region of thebeta-tubulingene sequence of the pathogen was targeted and the recombinase aided amplification (RAA) primers were designed. Lateral flow chromatographic strips were used to establish and further optimize a rapid detection system. The results showed that the developed RAA amplification could be completed within 30 minutes at a constant temperature of 39 ℃ and had high specificity with a detection limit of 100 fg/μL. Its sensitivity was comparable to that of conventional PCR detections. The optimal amplification time and temperature of the optimized detection system were 20-35 min and 31-39 ℃. This LFD-RAA detection system could be applied for rapid detection of the pathogen of tobacco target spot on tobacco plants.
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