PCR快速检测种子携带绿豆晕疫病菌方法的应用

Rapid detection of seed carrying Pseudomonas savastanoi pv.phaseolicola in mung beans using PCR

【目的】通过PCR快速检测方法筛选对种子表面携带绿豆晕疫病菌灵敏度较高的引物,为绿豆晕疫病菌的检测工作提供技术支持。【方法】选择7对特异性引物,对绿豆晕疫病菌不同浓度菌悬液进行一系列的PCR反应,并验证其灵敏度。【结果】最终筛选出灵敏度较高的3对引物;模拟种子表面带菌检测结果表明,引物B4-1-F/B4-1-R和B4-2-F/B4-2-R检测的最小菌悬液浓度均为1×10^(4) CFU/mL,引物HB14F/HB14R检测的最小菌悬液浓度为1×10^(6) CFU/mL;种子带菌率检测结果表明,引物B4-1-F/B4-1-R和B4-2-F/B4-2-R振荡洗涤1 h即可检出带菌率为0.0625%的种子样品,引物HB14F/HB14R振荡洗涤4 h后可检出带菌率为0.125%的种子样品。【结论】PCR快速检测方法可直接对种子表面携带绿豆晕疫病菌进行检测,方便快捷。

【Objective】Using PCR to rapidly screen primers with high sensitivity to the Pseudomonas savastanoi pv.phaseolicola on mung bean seed surface.Provide technical support for the detection of seed-carrying Pseudomonas savastanoi pv.phaseolicola in mung bean.【Methods】Seven pairs of specific primers were selected to perform a series of PCR reactions on Pseudomonas savastanoi pv.phaseolicola suspensions at different concentrations,and their sensitivity was verified.【Results】Three pairs of primers with high sensitivity were selected.The simulated seed surface bacteria detection results showed that the minimum concentration of bacterial suspension detected by primer B4-1-F/B4-1-R and B4-2-F/B4-2-R was 1×10^(4) CFU/mL,and the minimum concentration of bacterial suspension detected by primer HB14F/HB14R was 1×10^(6) CFU/mL.The results showed that primer B4-1-F/B4-1-R and B4-2-F/B4-2-R could detect the seed sample with seed-carrying rate of 0.0625%after shaking and washing for 1 h,and primer HB14F/HB14R could detect the seed sample with seed-carrying rate of 0.125%after shaking and washing for 4 h.【Conclusion】PCR could be used to rapidly detect seed carrying the Pseudomonas savastanoi pv.phaseolicola on mung bean seed surface,which was convenient and quick.