环介导等温扩增法可视化快速检测玉米中的黑曲霉

Visual rapid detection of Aspergillus niger in Zea mays L. by loop-mediated isothermal amplification

中文关键词:  黑曲霉  环介导等温扩增  赭曲霉毒素  可视化  快速检测

英文关键词:Aspergillus niger  loop-mediated isothermal amplification  ochratoxin  visualization  rapid detection

中文摘要:

      目的 建立环介导等温扩增技术(loop-mediated isothermal amplification, LAMP)快速可视化检测玉米中黑曲霉(Aspergillus niger)的方法。方法 基于LAMP技术, 针对赭曲霉毒素A (ochratoxin A, OTA)产毒关键基因OTAhal设计特异性引物, 通过对引物序列、引物比例、反应温度、目视染料等条件的优化, 确定最佳检测条件; 以黑曲霉总DNA和人工污染玉米样品为模板, 验证方法的检测灵敏度和实际应用的可行性。结果 最佳引物比例为8:4:1, 最佳反应温度为64 ℃, 方法可在30 min内完成目标DNA扩增, 阳性样本的颜色由紫罗兰色转变为蓝紫色。该方法可检测低至6.87×10–3 ng/μL的DNA质量浓度, 应用于玉米实际样品测定时, 可检测低至101个/mL的孢子浓度。结论 该方法操作简单、灵敏度高、无需大型仪器, 能应用于玉米实际样品中OTA的检测, 具有广阔的应用前景。

英文摘要:

      Objective To establish a rapid visual detection method for Aspergillus niger in Zea mays L. by loop-mediated isothermal amplification (LAMP) technology. Methods Based on LAMP technology, specific primers targeting the key ochratoxin A (OTA) biosynthesis gene OTAhal were designed. Through screening of the primers and optimization of conditions such as primer sequence, primer ratio, reaction temperature and visual dye, the optimal detection conditions were determined. Using the total DNA of Aspergillus niger and artificially contaminated corn samples as templates, the sensitivity of the detection method and the feasibility of its practical application were validated. Results The optimal primer ratio was 8:4:1, the optimal reaction temperature was 64 °C, and the method could complete the amplification of target DNA within 30 minutes. The color of the positive sample changed from violet to blue-purple. This method could detect DNA concentrations as low as 6.87×10?3 ng/μL. When applied to actual Zea mays L. sample testing, it achieved a detection limit as low as 101 spores/mL. Conclusion This method is simple to operate, highly sensitive, and does not require sophisticated instruments. It can be applied to the detection of OTA in actual Zea mays L. samples, indicating broad application prospects.