草莓白粉病菌LAMP可视化快速检测方法的建立及初步应用  

Establishment and Application of Visual LAMP Method for Rapid Detection of Sphaerotheca aphanis Causing Strawberry Powdery Mildew

作　　者：贲海燕 郝永娟[1] 霍建飞 姚玉荣 高苇 王万立[1] BEN Haiyan;HAO Yongjuan;HUO Jianfei;YAO Yurong;GAO Wei;WANG Wanli(Institute of Plant Protection,Tianjin Academy of Agricultural Sciences,Tianjin 300381,China)

机构地区：[1]天津市农业科学院植物保护研究所,天津300381

出　　处：《华北农学报》2022年第6期182-190,共9页Acta Agriculturae Boreali-Sinica

基　　金：天津市科技支撑项目(19YFZCSN00480)。

摘　　要：由羽衣草单囊壳菌引起的草莓白粉病是保护地草莓发病面积较大、发病频率较高的草莓病害之一,从苗期到结果期都能发生危害。建立快速、简便、高效的草莓白粉病病原菌检测技术对于该病的有效诊断和防控尤为重要。以羽衣草单囊壳菌ITS基因保守序列为靶基因,设计了1组包括F3/B3、FIP/BIP和LF/LB的环介导等温扩增(LAMP)特异性引物,通过实时荧光曲线和荧光显色双重判定方法,对体系中影响检测效率的主要因素dNTPs、MgSO_(4)、BstDNA聚合酶、甜菜碱及温度进行优化,并对优化后的方法进行特异性、灵敏性检测及田间样本检测效果评估。结果表明,建立了草莓白粉病菌LAMP检测体系后,其优化的dNTPs终浓度为1.6 mmol/L,MgSO_(4)终浓度为8 mmol/L,BstDNA聚合酶终浓度为320 U/moL,甜菜碱终浓度为1.2 mmol/L,反应最佳温度为65℃。该方法能够特异性地检测出草莓白粉病病原菌,检测灵敏度达到3.2×10^(-4)ng/μL,最低检测浓度可在1 h内完成,效率是普通PCR的100倍。通过LAMP荧光显色法进行草莓白粉病田间样品检测,可有效地检测草莓白粉病发病症状不明显的初侵染样本。该方法具有检测时间短、肉眼直接观察结果、对操作人员要求低、检测成本低等优点,对于草莓白粉病早期诊断、监测,提早预防、确定最佳防治时间具有重要的指导意义。Strawberry powdery mildew caused by Sphaerotheca aphanis is one of the strawberry diseases with large incidence area and high incidence frequency in the protected cultivation areas,which usually occurs from seedling stage to fruiting stage.Therefore,the establishment of a rapid and efficient detection method of S.aphanis is quite important for the diagnosis and control of strawberry powdery mildew.A set of LAMP amplification primers including F3/B3、FIP/BIP and LF/LB were designed for the conserved sequence of ITS gene of Sphaerotheca aphanis as the target gene.Through the double judgment method of Real-time fluorescence curve and fluorescence color change,the main factors in the LAMP reaction system including the final concentrations of dNTPs,MgSO_(4),BstDNA polymerase and betaine and amplification temperature were selected and optimized to increased etections ensitivity and specificity.Finally,the effects of field samples detection by optimized LAMP were evaluated.The results showed that the LAMP detection system of strawberry powdery mildew was established successfully.The optimized reaction conditions included dNTPs 1.6 mmol/L,MgSO_(4)8 mmol/L,BstDNA polymerase 320 U/moL and betaine 1.2 mmol/L and the amplification temperature was 65℃.Under such conditions,the minimum detection sensitivity was 3.2×10^(-4)ng/μL within 60 min,and the efficiency of which was 100 times higher than the result of PCR amplification.The visual LAMP method could effectively detect the samples with no obvious symptoms infected by S.aphanis in the early stage.This method has the advantages of short detection time,direct observation with eyes,low requirements for operators and low detection cost.It has important guiding significance for the early diagnosis,monitoring,early prevention and determination of the best control time of strawberry powdery mildew.

关 键 词：草莓白粉病 羽衣草单囊壳菌 环介导等温扩增检测技术(LAMP) 检测 

分 类 号：S436.68[农业科学—农业昆虫与害虫防治]