玉米内州萎蔫病菌荧光重组酶介导等温扩增检测方法的建立

玉米内州萎蔫病菌(Clavibacter michiganensis subsp. nebraskensis)是进境玉米重要检疫性病原物之一。通过分析比较玉米内州萎蔫病菌的纤维素酶基因celB,筛选出独特和保守的基因序列用于设计引物和探针,利用重组酶介导等温扩增技术(recombinase-aided amplification,RAA)建立玉米内州萎蔫病菌的荧光RAA检测方法。以celB基因序列作为靶标序列设计出引物2F、6R和探针P,建立了玉米内州萎蔫病菌荧光RAA检测方法,该方法在39 ℃恒温条件下,20 min内可完成检测反应,特异性好,且灵敏度高达130 fg·μL^-1,利用该方法检测4份玉米模拟样本阳性,8份玉米真实样品阴性,检测结果与参照国家标准GB/T 36840—2018一致。结果表明,建立的荧光RAA检测方法快速、特异、灵敏,能够满足现场检测要求。

Development of a fluorescent recombinase-aided amplification method for Clavibacter michiganensis subsp. nebraskensis detection

Clavibacter michiganensis subsp. nebraskensis(Cmn) is one of the important quarantine pathogens of imported corn. Our research objective was to develop a robust and rapid fluorescent recombinase-aided amplification (RAA) to detect Cmn. Comparative analyses of cellulase gene were performed to identify unique and conserved gene regions for primer and probe design. The unique celB sequence was determined for specific detection of Cmn. After primers screening and specificity verification, the fluorescent RAA assay with primers 2F/6R and probe P was established. Under 39 ℃, the fluorescent RAA could detect Cmn specifically with the sensitivity at 130 fg·μL^-1 in 20 min. Simulated samples and actual samples were used to evaluate the reliability of fluorescent RAA, the detection result was the same as GB/T 36840—2018. Overall, the fluorescent RAA assay established in this study was high speed, specificity, and high sensitivity, which is proved to be a reliable method for Cmn detection.