向日葵茎溃疡病菌RPA检测方法的建立

Recombinase polymerase amplification(RPA)for the detection of Diaporthe helianthi

向日葵茎溃疡病菌(Diaporthe helianthi)是我国进境植物检疫性病原真菌。本研究针对D.helianthi的cal基因保守序列设计特异性引物,建立该病菌的重组酶聚合酶扩增检测技术(RPA)方法,并对其特异性、灵敏度及适用性进行评价。结果显示,建立的RPA方法特异性强,只有3个D.helianthi样品能够检测到234 bp的目的片段;方法灵敏度达到0.1 ng/μL;从模拟带菌的种子中也能够成功检测到目标扩增片段。本研究建立D.helianthi的RPA检测方法具有较高的特异性和灵敏度,能够直接应用于种子带菌检测,适用于口岸进境向日葵籽的现场快速检测。

Diaporthe helianthi is an aggressive pathogen causing sunflower stem canker disease,which has been listed as a quarantine fungus by China.In this research,the recombinase polymerase amplification(RPA)assay was developed for the detection of D.helianthi,and a pair of specific primers for RPA were designed based on the cal gene of D.helianthi,and the specificity and sensitivity of the assay were tested.The results showed that the assay was specific,target fragments of 234 bp were observed only in 3 D.helianthi samples.The detection limit of this assay was 0.1 ng/μL genomic DNA,and amplified fragments 234 bp were observed from seed simulated infection with Diaporthe helianthi.In conclusion,the RPA assay is high specific and sensitive,and is suitable for on-site rapid detection of imported sunflower seeds.