利用qRT-PCR技术对PVY重组株系的鉴定

Identification of Recombinant Strain PVY by qRT-PCR Technique

【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PVY^(N-Wi)和PVY^(NTN-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Gen Bank已公布的PVY^(N-Wi)和PVY^(NTN-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PVY^(N-Wi)和PVY^(NTN-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PVY^(N-Wi)株系侵染;27份样品,受到PVY^(NTN-NW)株系侵染;同时存在9份样品,可能同时受到PVY^(N-Wi)、PVY^(NTN-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 PVY^（N-Wi）and PVY^（NTN-NW）strains 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 PVY^（N-Wi）and PVY^（NTN-NW）strains published in Gen Bank,and real-time quantitative PCR（ qRT-PCR） was used for detection.【Results】The PVY^（N-Wi）and PVY^（NTN-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 PVY^（N-Wi） strain,27 samples were infected by PVY^（NTN-NW）strain,and there were 9 samples,the infection might be affected by PVY^（N-Wi）and PVY^（NTN-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.