多重PCR技术检测4种大豆镰孢菌根腐病病原

Establishment of a multiplex PCR for detection of four Fusarium pathogens of soybean root rot disease

镰孢菌是引起大豆根腐病的常见病原菌,传统检测病原镰孢菌的方法存在操作繁琐、时间长、成本高和效率低等缺点,建立一种快速检测方法显得尤为重要。本研究基于翻译延伸因子序列(EF-1α)建立了检测镰孢菌(Fusarium species)的多重PCR反应体系,检测了多重PCR体系灵敏度,模拟侵染样本验证多重PCR技术的可行性。实验结果表明,该方法能够通过扩增片段的大小区分锐顶镰孢菌(Fusarium acuminatum)、尖孢镰刀菌(Fusarium oxysporum)、茄病镰孢菌(Fusarium solani)和禾谷镰孢菌(Fusarium graminearum)。灵敏度检测显示,最低检测基因组DNA浓度达1×10^(-4)ng/μL;模拟侵染样本实验表明,使用镰孢菌和大豆基因组混合样本作为模板,在DNA浓度为100 ng/μL时仍能准确检测出目的条带。因此。以翻译延伸因子序列为靶标建立的多重PCR技术,能够灵敏、特异性地检测出该四种镰孢菌,可在复合侵染引起大豆镰孢菌根腐病的病原菌鉴定中准确检测。

Fusariumspecies are the most common pathogens of soybean root rot disease. Traditional detection methods for Fusarium are complicated, time and money comsumming with low efficiency. It’s particularly important to establish a rapid detection method for detection of soybean Fusarium root rot disease. In our research, a multiplex PCR system was established based on the translation elongation factor gene(EF-1α). The sensitivity of the multiplex PCR reaction system and viability to detect simulating infection soybean tissues were carried out, the lowest concentration of genome DNA is 1×10^(-4) ng/μL. We successfully detected Fusarium acuminutum, Fusarium oxysporum, Fusarium solani and Fusarium graminearum with the multiplex PCR detection system. The EF-1α bands were successfully amplified with genomes DNA of 100 ng/μL of the 4 Fusarium species or simulating infection samples as templates. In conclusion, a multiplex PCR detection method based on translation elongation factor gene was established which worked well to distinguish 4 different Fusarium species, the method could be used to diagnose Fusarium pathogens in multiple infection.