Su Yachun, Yang Yuting, Peng Qiong, Zhou Dinggang, Chen Yun, Wang Zhuqing, Xu Liping, Que Youxiong
Key Laboratory of Sugarcane Biology and Genetic Breeding, Fujian Agriculture and Forestry University, Ministry of Agriculture, Fuzhou 350002, China.
Sci Rep. 2016 Apr 1;6:23994. doi: 10.1038/srep23994.
Smut is a fungal disease with widespread prevalence in sugarcane planting areas. Early detection and proper identification of Sporisorium scitamineum are essential in smut management practices. In the present study, four specific primers targeting the core effector Pep1 gene of S. scitamineum were designed. Optimal concentrations of Mg(2+), primer and Bst DNA polymerase, the three important components of the loop-mediated isothermal amplification (LAMP) reaction system, were screened using a single factor experiment method and the L16(4(5)) orthogonal experimental design. Hence, a LAMP system suitable for detection of S. scitamineum was established. High specificity of the LAMP method was confirmed by the assay of S. scitamineum, Fusarium moniliforme, Pestalotia ginkgo, Helminthospcrium sacchari, Fusarium oxysporum and endophytes of Yacheng05-179 and ROC22. The sensitivity of the LAMP method was equal to that of the conventional PCR targeting Pep1 gene and was 100 times higher than that of the conventional PCR assay targeting bE gene in S. scitamineum. The results suggest that this novel LAMP system has strong specificity and high sensitivity. This method not only provides technological support for the epidemic monitoring of sugarcane smut, but also provides a good case for development of similar detection technology for other plant pathogens.
黑穗病是一种在甘蔗种植区广泛流行的真菌病害。早期检测和正确鉴定甘蔗黑粉菌对于黑穗病的防治至关重要。在本研究中，设计了针对甘蔗黑粉菌核心效应蛋白Pep1基因的4条特异性引物。采用单因素试验方法和L16(4(5))正交试验设计，对环介导等温扩增（LAMP）反应体系的3个重要组分Mg(2+)、引物和Bst DNA聚合酶的最佳浓度进行了筛选。由此，建立了一种适用于检测甘蔗黑粉菌的LAMP体系。通过对甘蔗黑粉菌、串珠镰刀菌、银杏拟盘多毛孢、甘蔗长蠕孢、尖孢镰刀菌以及崖城05-179和新台糖22号内生菌的检测，证实了LAMP方法具有高特异性。LAMP方法的灵敏度与针对Pep1基因的常规PCR相同，比针对甘蔗黑粉菌bE基因的常规PCR检测高100倍。结果表明，这种新型LAMP体系具有很强的特异性和高灵敏度。该方法不仅为甘蔗黑穗病的流行监测提供了技术支持，也为开发其他植物病原菌的类似检测技术提供了良好范例。
