基于酶介导双重指数扩增检测技术的白壳快速检测方法  

Detection of Albonectria rigidiuscula based on enzyme mediated duplex exponential amplification

作　　者：俞紫琳 张慧丽 顾建锋 吴尧 胡振新 李秋实 段维军 YU Zilin;ZHANG Huili;GU Jianfeng;WU Yao;HU Zhenxin;LI Qiushi;DUAN Weijun(Ningbo Academy of Inspection and Quarantine,Ningbo 315012,Zhejiang,China;Ningbo Joysun Product Testing Service Company,Ningbo 315012,Zhejiang,China;Technical Center of Ningbo Customs District P.R.China,Ningbo 315012,Zhejiang,China;GeneVide Biotech Co.,Ltd.,Suzhou 215000,Jiangsu,China)

机构地区：[1]宁波检验检疫科学技术研究院,浙江宁波315012 [2]宁波中盛产品检测有限公司,浙江宁波315012 [3]宁波海关技术中心,浙江宁波315012 [4]苏州晶睿生物科技有限公司,江苏苏州215000

出　　处：《菌物学报》2024年第9期105-113,共9页Mycosystema

基　　金：国家重点研发计划(2022YFF0608804);海关总署科研项目(2022HK011);宁波市社会公益科研项目(2022S010)。

摘　　要：可可花瘿病致病菌白壳Albonectria rigidiuscula是我国进境植物检疫性有害生物,已在多个口岸植物入境产品中被截获,存在较大生物入侵风险。为探索口岸适用的现场快速检测技术,根据白壳翻译延长因子TEF1-α的基因序列,基于酶介导双重指数扩增核酸检测技术设计了针对白壳的5条DNA上游引物、6条DNA下游引物和4条RNA探针。通过筛选,获得了扩增效果最佳的上、下游DNA引物和RNA探针组合。灵敏度实验结果表明,其最低检测限量可达0.5 pg/uL。实际样品检测结果表明,该方法适用于口岸疑似携带有白壳样品的检测与初筛。该方法具有操作简便、耗时较短、特异性强等优点,整个反应过程仅需30 min,检测过程完全闭管且不需要PCR后续处理,为口岸现场白壳检测筛查提供了重要参考。Albonectria rigidiuscula is listed as Chinese quarantine pest,and has been intercepted at many ports in China,which may cause invasion risks.A precise enzyme mediated duplex exponential amplification(EmDEA)assay was developed for detection of A.rigidiuscula.Based on translation elongation factor(TEF1-α)gene of A.rigidiuscula and related species,five upstream DNA primers,six downstream DNA primers,and four RNA probes were designed and synthesized.The minimum detection limit is up to 0.5 pg/uL.The detection results of suspected sample indicate this method is useful to detect and screen A.rigidiuscula.The method is rapid,sensitive and completed within a single tube,without post-PCR handling of the amplification products.The entire reaction process takes about 30 minutes.This result provides an important reference for the detection and screening of A.rigidiuscula in the port.

关 键 词：外来物种 生物安全 植物病害 检疫 检测 

分 类 号：S41[农业科学—植物保护]