木尔坦棉花曲叶病毒RPA快速检测方法的建立

Establishment of RPA for rapid detection of Cotton leaf curl Multan virus

木尔坦棉花曲叶病毒Cotton leaf curl Multan virus(CLCuMuV)引起的病害是世界棉花生产上的毁灭性灾害,也是我国进境植物检疫性有害生物之一。因此,建立快速检测技术对CLCuMuV的检疫和防控具有重要意义。本研究根据CLCuMuV外壳蛋白(CP)基因序列设计引物,建立了该病毒的重组酶聚合酶等温扩增(recombinase polymerase amplification,RPA)检测方法,并评价了该方法的灵敏度和特异性,进一步测定了对田间疑似病样的检测准确性。结果表明,建立的RPA检测方法仅能从感染CLCuMuV的样品中扩增出目的条带,而感染同属的其他5种病毒的样品中未扩增出目的条带。该方法检测灵敏度是常规PCR的10倍,且对田间疑似病样的检出结果与PCR试验结果一致。因此,本研究所建立的CLCuMuV RPA快速检测方法具有特异、灵敏、准确、操作简便、无需特殊设备等优点,这些为CLCuMuV的快速检测提供了一种新技术。

Cotton leaf curl Multan virus(CLCuMuV)causes destructive diseases in cotton crops worldwide,which is a quarantine organism subjected to phytosanitary regulations in China.The rapid detection technique is very important for the quarantine and control of CLCuMuV.In this study,a recombinase polymerase amplification(RPA)detection method was established based on the coat protein(CP)gene sequence of CLCuMuV.The sensitivity and specificity of this method were evaluated.The accuracy of this method was determined by detecting the suspected samples in fields.The results showed that the established RPA detection method amplified the target fragment only from CLCuMuV-infected samples,and no fragment was amplified from the samples infected by other five viruses belonging to the same genus with CLCuMuV.The sensitivity of the established RPA detection method was 10-fold higher than that of the conventional PCR.The RPA detection results of suspected diseased samples in fields were consistent with the PCR results.Therefore,the rapid CLCuMuV RPA detection method was established in this study,characterized by high specificity,sensitivity,accuracy,easiness to operate,and no requirement for special equipment.It provides a new technique for rapid detection of CLCuMuV.