百合PlAMV和LMoV双重RT-PCR检测体系的建立  

Establishment of Duplex RT-PCR Detection System for PlAMV and LMoV in Lily(Lilium spp.)

作　　者：王丽花[1] 杨秀梅[1] 何晓琴 张艺萍[1] 段青[1] 杨舒淇 苏艳[1] WANG Li-Hua;YANG Xiu-Mei;HE Xiao-Qin;ZHANG Yi-Ping;DUAN Qing;YANG Shu-Qi;SU Yan(Flower Research Institute/Yunnan Key Laboratory for Flower Breeding/National Engineering Research Center for Ornamental Horticulture,Yunnan Academy of Agriculture Science,Kunming 650205,China;Qingyun subdistrict office Panlong District Government,Kunming 650051,China)

机构地区：[1]云南省农业科学院花卉研究所/云南省花卉育种重点实验室/国家观赏园艺工程技术研究中心,昆明650205 [2]昆明市盘龙区人民政府青云街道办事处,昆明650051

出　　处：《农业生物技术学报》2025年第3期670-679,共10页Journal of Agricultural Biotechnology

基　　金：云南省创新引导与科技型企业培育计划(202204BI090022);云南省种子种业联合实验室项目(202205AR070001);国家花卉产业体系昆明综合试验站项目(CARS-23-G56)。

摘　　要：车前草花叶病毒(Plantago asiatica mosaic virus, PlAMV)和百合斑驳病毒(Lily mottle virus, LMoV)是目前对我国百合(Lilium spp.)产业危害最严重的2种病毒。为建立上述2种病毒的快速高效检测体系,本研究首先提取百合感病植株总RNA,经逆转录获得cDNA,后以cDNA为模板开展双重同步检测体系优化,并对其特异性和灵敏度等进行检测。结果表明,建立的双重同步检测体系对PlAMV和LMoV的特异性扩增片段大小分别为910和521 bp,对其他常见病毒均无扩增,表明所建立方法特异性强;PlAMV和LMoV的最低检出限分别为967.5 fg和59.0 pg,相比于单基因RT-PCR检测的最低检出限(PlAMV 19.35fg, LMoV 11.8 pg),灵敏度分别降低了50倍和5倍;扩增产物测序片段与不同来源的19个PlAMV分离物和21个LMoV分离物开展BLAST序列分析显示,PlAMV的相似性为86.71%~99.40%,LMoV的相似性为94.75%~99.49%。利用双重同步检测体系对19个田间样品进行检测应用,结果显示,19个样品中有3个样品同时携带PlAMV和LMoV,2个样品携带PlAMV,4个样品携带LMoV,其余10个样品未检出,该结果与基因芯片法检测结果一致。综上所述,本研究所建立的检测体系可同时高效检测PlAMV和LMoV2种病毒,结果可靠,应用前景广阔。The plantago asiatica mosaic virus(PlAMV)and the Lily mottle virus(LMoV)are currently the two most serious viral pathogens affecting the lily(Lilium spp.)industry in China.The aim of this study is to develop a rapid and efficient detection system for these 2 viruses.Firstly,total RNA was extracted from diseased lily plants,and cDNA was obtained through reverse transcription.Subsequently,a dual-synchronous detection system was optimised using cDNA as a template,and the specificity and sensitivity of the detection system were evaluated.The results demonstrated that the established dual-synchronous detection system amplified specific segments of 910 and 521 bp for PlAMV and LMoV,respectively,with no amplification for other common viruses,indicating strong detection specificity.The lowest detection limits for PlAMV and LMoV were 967.5 fg and 59.0 pg,respectively,which were 50 times and 5 times lower than the lowest detection limits of single-gene RT-PCR detection(PlAMV 19.35 fg,LMoV 11.8 pg).Sequence analysis of the amplified products with sequencing segments from 19 PlAMV isolates and 21 LMoV isolates from different sources revealed similarities of 86.71%to 99.40%for PlAMV and 94.75%to 99.49%for LMoV.The dual-synchronous detection system was employed to examine 19 field samples,and the results indicated that 3 samples were positive for both PlAMV and LMoV,2 samples were positive for PlAMV,4 samples were positive for LMoV,and the remaining 10 samples were not detected,which was consistent with the results of gene chip detection.In conclusion,the detection system this study established is capable of detecting both PlAMV and LMoV viruses simultaneously,with reliable results and broad application prospects.

关 键 词：百合 双重RT-PCR 车前草花叶病毒(PlAMV) 百合斑驳病毒(LMoV) 

分 类 号：S436.8[农业科学—农业昆虫与害虫防治]