马铃薯腐烂茎线虫RPA-LFD可视化快速检测方法的建立

Development of RPA-LFD visualization assay for rapid detection of Ditylenchus destructor

马铃薯腐烂茎线虫是为害我国甘薯和马铃薯的一种重要植物病原线虫,也是我国重要的检疫性有害生物。为实现对该线虫的准确、快速且可视化的检测,本研究以马铃薯腐烂茎线虫rDNA-ITS序列为靶标构建了重组酶聚合酶结合侧流层析试纸条(RPA-LFD)的可视化快速检测体系。该体系可在39℃条件下15 min内特异性地完成对马铃薯腐烂茎线虫的检测,对A型(甘薯种群)和B型(马铃薯种群)单头线虫(J4)的检出底限均为3125-1头线虫,可以直接对土壤和甘薯茎中的马铃薯腐烂茎线虫进行检测,灵敏度可达1头(J4)/10 g土壤和1头(J4)/2 g甘薯茎组织。该体系操作简便、成本低廉、结果可视,可为马铃薯腐烂茎线虫的早期预警和口岸检疫提供技术支撑。

Ditylenchus destructor is an important plant pathogenic nematode of sweet potato and potato,and also an important quarantine pest in China.To achieve accurate,rapid and visual detection of this nematode,the rDNA-ITS sequence of D.destructor was used as the target to construct the recombinase polymerase amplification combined with a lateral flow dipstick(RPA-LFD)visual rapid detection assay.The assay can detect D.destructor specifically within 15 min at 39℃,and the detection limit for a single nematode(J4)of type A(sweet potato population)and type B(potato population)is 3125-1nematodes.It can directly detect D.destructor in soil and sweet potato stem tissue,with a sensitivity of one J4 nematode per 10 g soil and one J4 nematode per 2 g plant tissue.In addition,the assay also has the advantages of simple operation,low cost and visual result.The assay will lay a foundation for the early warning and port quarantine of D.destructor.