3种甘薯病毒多重PCR检测方法的建立与应用  

Establishment and Application of Multiplex PCR for Detection of Three Viruses in Sweet Potato

作　　者：李文宗 梁鑫 杨露露 王润豪 王磊[2] LI Wen-zong;LIANG Xin;YANG Lu-lu(Henan Huazhi Nutrition Technology Co.,Ltd.,Xinxiang,Henan 453000)

机构地区：[1]河南华智营养科技有限公司,河南新乡453000 [2]中国农业科学院生物技术研究所,北京100081

出　　处：《安徽农业科学》2022年第22期89-93,126,共6页Journal of Anhui Agricultural Sciences

摘　　要：针对甘薯卷叶病毒(SPLCV)、甘薯羽状斑驳病毒(SPFMV)、甘薯褪绿矮化病毒(SPCSV)的外壳蛋白(CP)基因序列,设计了3种病毒的3对特异性引物,扩增大小分别为180、295、774 bp,通过对引物添加量、退火温度、循环数等反应条件的优化,完善了能同时扩增出SPLCV、SPFMV、SPCSV 3种病毒的多重PCR体系,可以实现同时对甘薯3种病毒104拷贝水平的检测,并具有良好的特异性。利用该技术体系,对不同田间样品进行了检测,PCR产物测序表明,3种病毒序列与已知的参考序列同源率一致性分别达到94%、94%、97%及以上。建立能够同时进行3种病毒检测的多重PCR检测技术体系,可以快速准确地进行甘薯脱毒苗病毒检测和田间样品的诊断。Three pairs of specific primers for sweet potato leaf curl virus(SPLCV),sweet potato feathery mottle virus(SPFMV)and sweet potato chlorotic stunt virus(SPCSV)were designed based on conserved region sequences of the coat protein(CP)genes published in NCBI.The amplification size was 180,295 and 774 bp respectively.By optimizing the reaction conditions such as primer addition amount,annealing temperature and number of cycles,the multiplex PCR system that could simultaneously amplify three viruses,namely,SPLCV,SPFMV and SPCSV were improved.It could simultaneously detect 104 copy levels of three sweet potato viruses,and had good specificity.Using this method to detect field samples,the compound infected virus could be detected accurately.Sequence analysis showed that the sequence homology of the target fragments were 98%,94% and 97% respectively.The results showed that the establishment of a multiplex PCR detection technology system capable of simultaneously detecting three viruses could rapidly and accurately conduct virus detection of sweet potato virus-free seedlings and diagnosis of field samples.

关 键 词：甘薯 甘薯卷叶病毒(SPLCV) 甘薯羽状斑驳病毒(SPFMV) 甘薯褪绿矮化病毒(SPCSV) 多重PCR 

分 类 号：S435.313[农业科学—农业昆虫与害虫防治]