摘要
为了建立苹果牛眼果腐病原多年明孢盘菌Neofabraea perennans微滴式数字PCR (ddPCR)检测方法,实现苹果检疫性病原菌的高效率、高精度的检疫鉴定和疫情监控,为口岸外来有害生物防控和保护我国生态安全提供技术服务和支持。本研究以N.perennans为研究对象,通过生物信息学分析确定β-微管蛋白基因(TUB2)作为靶标进行特异性引物与探针设计,通过引物和探针浓度以及退火温度的优化,构建N.perennans的ddPCR特异性检测体系。研究结果表明,N.perennans的ddPCR检测体系的绝对定量检测低限为0.16 copies/μL,最低基因组DNA检测浓度为10.0 pg/μL,与qPCR方法一致,在定量检测范围内的线性相关系数R2大于0.999,RSD小于25%,说明该检测体系的重复性较好,能够用于进口水果样品中苹果牛眼果腐病原多年明孢盘菌N.perennans的快速精准检测。
In order to realize high efficiency and high precision quarantine identification and epidemic monitoring of apple quarantine pathogen,the detection method of Neofabraea perennans by droplet digital PCR(ddPCR)was established.This method provides technical services and support for the prevention of pest from ports and the protection of China’s ecological security.N.perennans was used as the research object,and the β-tubulin gene(TUB2)was identified by bioinformatics analysis as a target for specific primer and probe design.The specific detection system of N.perennans by ddPCR was established by optimizing the concentration of primers,probes and annealing temperature.The results showed that the detection limit of ddPCR was 0.16 copies/μL and the detection concentration of genomic DNA was 10.0 pg/μL,being consistent with the result of qPCR.The linear correlation coefficient in the range of quantitative detection was more than 0.999,and the RSD was less than 25%,indicating that the detection system had good repeatability and could be used for rapid and accurate detection of N.perennans in imported fruit samples.
作者
李辉
姜锋
杨春光
姜丽
赵鹏
李鑫
LI Hui;JIANG Feng;YANG Chunguang;JIANG Li;ZHAO Peng;LI Xin(Technical Center of Dalian Customs,Dalian 116000,Liaoning,China;Integrated Technical Service Center of Donggang Customs,Dandong 118300,Liaoning,China;College of Humanities&Information,Changchun University of Technology,Changchun 130122,Jilin,China;State Key Laboratory of Mycology,Institute of Microbiology,Chinese Academy of Sciences,Beijing 100101,China)
出处
《菌物学报》
CAS
CSCD
北大核心
2024年第9期67-79,共13页
Mycosystema
基金
“十四五”国家重点研发计划(2021YFD1400100)。
