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象耳豆根结线虫(Meloidogyne enterolobii)是一种具有高致病力的植物寄生线虫，是欧洲和地中海植物保护组织、美国等多个国家和地区的检疫性有害生物。本研究以象耳豆根结线虫的ITS序列为靶标，设计RPA引物和EXO荧光检测探针。经反应条件优化、特异性及灵敏度验证，建立了象耳豆根结线虫的RPA-EXO荧光检测方法。结果表明，象耳豆根结线虫RPA-EXO检测体系的最佳反应条件为39℃、30 min,3个象耳豆根结线虫群体均有扩增曲线，其他6种线虫样品和空白对照均未见扩增曲线，说明引物探针具有良好的特异性。RPA-EXO检测灵敏度可达10-4头雌虫DNA。因此，本方法具有特异性强、灵敏度高、反应快速等特点，适用于基层实验室开展象耳豆根结线虫的检测。
Abstract：Meloidogyne enterolobii is a plant-parasitic nematode with high pathogenicity and is a quarantine pest in many countries and regions,including the European and Mediterranean Plant Protection Organization(EPPO) and the United States. In this study,the ITS sequence of Meloidogyne enterolobii was used as the target to design RPA primers and EXO fluorescent probes. Through the optimization of reaction conditions,as well as the verification of specificity and sensitivity,an RPA-EXO fluorescent detection method for Meloidogyne enterolobii was established. The results showed that the optimal reaction conditions for the RPA-EXO detection system of Meloidogyne enterolobii were 39℃ for 30 minutes. Amplification curves were obtained for the three populations of Meloidogyne enterolobii,while no amplification curves were observed for the samples of the other six species of nematodes and the blank control,indicating that the primer and probe had good specificity. The detection sensitivity of RPA-EXO could reach the 10-4 of single female. Therefore,this assay is characterized by high specificity,excellent sensitivity,and rapid response. It is suitable for the detection of Meloidogyne enterolobii in grassroots laboratories.
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基本信息:
DOI：10.19662/j.cnki.issn1005-2755.2025.03.006
中图分类号:S41-30
引用信息:
[1]冯自洋,焦莉苹,葛建军等.象耳豆根结线虫RPA-EXO荧光检测方法的建立[J].植物检疫,2025,39(03):35-40.DOI:10.19662/j.cnki.issn1005-2755.2025.03.006.
基金信息:
海南省科技专项资助重点研发项目（ZDYF2023XDNY079）