中国烟草科学
中國煙草科學
중국연초과학
CHINESE TOBACCO SCIENCE
2013年
3期
105-109
,共5页
宋丽云%杨金广%李锡宏%宋玉川%张长华%战徊旭%申莉莉%钱玉梅%王凤龙
宋麗雲%楊金廣%李錫宏%宋玉川%張長華%戰佪旭%申莉莉%錢玉梅%王鳳龍
송려운%양금엄%리석굉%송옥천%장장화%전회욱%신리리%전옥매%왕봉룡
免疫捕捉real-time PCR%黄瓜花叶病毒(CMV)%蚜虫
免疫捕捉real-time PCR%黃瓜花葉病毒(CMV)%蚜蟲
면역포착real-time PCR%황과화협병독(CMV)%아충
immunocapture real-time RT-PCR%Cucumber mosaic virus (CMV)%myzus persicae
由黄瓜花叶病毒（Cucumber mosaic virus, CMV）引起的病毒病是烟草上最主要的病害之一，高虫口密度的带毒蚜虫的迁入是烟草上CMV病害发生的主要致病因子。本研究结合当前CMV的两个检测体系——ELISA和real-time RT-PCR，通过CMV抗血清与带毒蚜虫研磨液中的病毒粒体结合反应，病毒复合体粘附在PCR管壁上，然后直接进行反转录反应，随后进行real-time PCR检测，建立了免疫捕捉real-time RT-PCR（Immunocapture real-time RT-PCR）检测单头蚜虫体内CMV的实时定量检测技术，与普通real-time RT-PCR和ELISA相比，灵敏性和特异性显著提高。该方法无需RNA提取等步骤，可以有效地减少样品操作过程的污染和蛋白、多糖以及酚类物质等杂质的影响，能够对微量CMV病毒粒体进行准确、快速、特异和灵敏的定量检测，对烟草病毒病的预测预报、无毒种苗的生产和病害防治都具有重要的科学意义。
由黃瓜花葉病毒（Cucumber mosaic virus, CMV）引起的病毒病是煙草上最主要的病害之一，高蟲口密度的帶毒蚜蟲的遷入是煙草上CMV病害髮生的主要緻病因子。本研究結閤噹前CMV的兩箇檢測體繫——ELISA和real-time RT-PCR，通過CMV抗血清與帶毒蚜蟲研磨液中的病毒粒體結閤反應，病毒複閤體粘附在PCR管壁上，然後直接進行反轉錄反應，隨後進行real-time PCR檢測，建立瞭免疫捕捉real-time RT-PCR（Immunocapture real-time RT-PCR）檢測單頭蚜蟲體內CMV的實時定量檢測技術，與普通real-time RT-PCR和ELISA相比，靈敏性和特異性顯著提高。該方法無需RNA提取等步驟，可以有效地減少樣品操作過程的汙染和蛋白、多糖以及酚類物質等雜質的影響，能夠對微量CMV病毒粒體進行準確、快速、特異和靈敏的定量檢測，對煙草病毒病的預測預報、無毒種苗的生產和病害防治都具有重要的科學意義。
유황과화협병독（Cucumber mosaic virus, CMV）인기적병독병시연초상최주요적병해지일，고충구밀도적대독아충적천입시연초상CMV병해발생적주요치병인자。본연구결합당전CMV적량개검측체계——ELISA화real-time RT-PCR，통과CMV항혈청여대독아충연마액중적병독립체결합반응，병독복합체점부재PCR관벽상，연후직접진행반전록반응，수후진행real-time PCR검측，건립료면역포착real-time RT-PCR（Immunocapture real-time RT-PCR）검측단두아충체내CMV적실시정량검측기술，여보통real-time RT-PCR화ELISA상비，령민성화특이성현저제고。해방법무수RNA제취등보취，가이유효지감소양품조작과정적오염화단백、다당이급분류물질등잡질적영향，능구대미량CMV병독립체진행준학、쾌속、특이화령민적정량검측，대연초병독병적예측예보、무독충묘적생산화병해방치도구유중요적과학의의。
Cucumber mosaic virus(CMV)is transmitted in nonpersistent manner by peach aphid (Myzus persicae), leading to many plant diseases. The large number of viruliferous peach M. persicae is one of the major culprits for the rampant outbreak of CMV disease in tobacco. In present study, Immunocapture real-time RT-PCR (IC-real-time RT-PCR) was investigated to detect CMV in individual peach aphid (M. persicae) by combining Enzyme-Linked Immunosorbent Assay(ELISA) and conventional RT-PCR. The complex of CMV CP antibodies and CMV particles was adhered to PCR tubes wall, then reverse transcriptase reaction was conducted directly, subsequently real-time PCR. Compared with conventional RT-PCR and ELISA to detect CMV in individual peach aphid, this method was more sensitive and specific. This assay of RT-PCR could be directly carried out without total RNA extraction by capturing CMV particle through CMV CP antibodies, and effectively reduce the pollution and inhibiting effects of proteins, polysaccharide and phenols materials in operating process. Therefore, as a specific, rapid, sensitivity and simple method for detecting slight amount CMV particle, IC-RT-PCR has significance of prediction, forecast and prevention of tobacco virus diseases.
