利用qRT-PCR技术对PVY重组株系的鉴定

摘要

[目的]马铃薯Y病毒(Potato virus Y,PVY)作为制约马铃薯生产的重要病毒之一,株系分化繁杂.近年被科学家研究分离的PVYN-Wi和PVYNTN-NW株系而言,其基因组结构极为相似但致病性差异较大.[方法]本研究利用qRT-PCR方法,方便快捷的检测出马铃薯Y病毒的这2种不同株系,同时并使之可成功且稳定的应用于马铃薯生产中.通过查找GenBank已公布的PVYN-Wi和PVYNTN-NW株系的全基因组序列,设计用于qRT-PCR检测,扩增目的片段大小为150 bP左右的特异性引物,应用实时荧光定量PCR技术(qRT-PCR)进行检测.[结果]通过设计的引物对保存的试管苗毒源进行检测,可以成功的精准鉴别出PVYN-Wi和PVYNTN-NW株系;同时对100份田间采集的叶片样品进行检测,成功的检测出100份样品中32份样品,受到PVYN-Wi株系侵染;27份样品,受到PVYNTN-NW株系侵染;同时存在9份样品,可能同时受到PVYN-Wi、PVYNTN-NW株系的侵染.[结论]实时荧光定量PCR技术(qRT-PCR)可适用于马铃薯叶片样品的检测,为株系的快速鉴定提供了可靠的方法,可用于生产中大规模样品的检测,有利于及时采取防控措施,降低损失.%[Objective] Potato virus Y (PVY) is one of the most important plant viruses that affect potato production,and its strain differentiation is complex.In particular,the genome structure is very similar to that of the PVYN-Wiand PVYNTN-NWstrains isolated by scientists in recent years,however,their pathogenicities are quite different.[Methods]In this study,the qRT-PCR method was used to detect and identify the two different strains of potato virus Y conveniently and quickly,and theoretical support was provided for the detection,prevention and control of samples in future production.Specific primers were designed for qRT-PCR detection,the amplification of the target fragment with a size of approximately 150 bp was determined through the complete genome sequence of the PVYN-Wi and PVYNTN-NW strains published in GenBank,and real-time quantitative PCR(qRT-PCR) was used for detection.[Results] The PVYN-Wi and PVYNTN-NW strains could be successfully and accurately identified through the design of the primers to the virus of the preservative known test-tube plantlet.At the same time,100 leaf samples collected in the field were analyzed,and it was successfully detected that 32 samples were infected by PVYN-Wi strain,27 samples were infected by PVYNTN-NWstrain,and there were 9 samples,the infection might be affected by PVYN-Wiand PVYNTN-NW strains in 100 samples.[Conclusion] Real-time quantitative PCR(qRT-PCR) could be applied for detection in potato leaf samples,providing a reliable method for the rapid identification of strains.Furthermore,it could be used for the detection of large-scale samples in production,which would be helpful for conducting timely and preventive measures to reduce loss.