烟草PVY Real-Time PCR 定量检测体系的建立及应用

刘晓霞; 杨金广; 王凤龙; 钱玉梅; 申莉莉; 张帅; 王长栓; 黄瑾

烟草PVY Real-Time PCR 定量检测体系的建立及应用

1.
国家烟草专卖局烟草病虫害监测与综合治理重点开放实验室,中国农业科学院烟草研究所,青岛 266101
2.
中国农业科学院研究生院,北京100081
3.
广西壮族自治区烟草公司百色市分公司,广西百色 533000

详细信息

作者简介:
刘晓霞，女，在读硕士，主要从事烟草病毒学研究。E-mail：happygirlnan@sina.com

通讯作者:
王凤龙

中图分类号: S435.72
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出版历程
- 收稿日期: 2009-07-23
- 刊出日期: 2010-02-27

Establishment and Application of Real-time PCR for Detection of Potato Virus Y in Tobacco

1.
Key Laboratory of Tobacco Pest Monitoring & Integrated Management, State Tobacco Monopoly Bureau, Tobacco Research Institute of CAAS, Qingdao 266101, China
2.
Graduate School of CAAS, Beijing 100081, China
3.
Baise Branch, Guangxi Tobacco Corporation, Baise, Guangxi 533000, China

摘要

摘要: 马铃薯Y 病毒是近年来危害烟草生产的重要病毒之一，严重影响烟草的产量与品质。本研究利用DNAMAN 软件对GenBank 数据库中已登录的马铃薯Y 病毒 (Potato virus Y, PVY) 全基因组序列进行序列比对，设计引物，以烟草肌动蛋白基因为内参，建立了烟草PVY 的实时定量检测体系。获得的real-time PCR 扩增基线平整，指数扩增明显，斜率大；稳定性和重现性好，变异系数小；循环阈值与PCR 起始模板量对数之间存在良好的线性关系。与DAS-ELISA 相比，该方法具有高效、灵敏、特异性强等优点，为从分子生物学水平上检测烟草中PVY 提供了新的技术手段。
- Real-time PCR /
- PVY /
- 检测
Abstract: Potato virus Y (PVY) is one of the most important viruses with huge damage to tobacco yields and quality in recent years. A quantitative method using real-time PCR technique basing on fluorescence dye SYBR Green was employed to detect PVY in tobacco in this study. PVY genome sequence alignments logged in Genebank were analyzed using DNAMAN software. Primer 5.0 software was used to design specific primers of PVY gene and actin gene. Results showed that the amplification curve had flat baseline, distinct exponential area, large and stable slope, and the coefficient of variation was very small. There was a linear relationship between threshold cycle values where samples crossed threshold and the logarithmic values of template concentration. Compared with DAS-ELISA, real-time PCR can be used as a new method to detect PVY in tobacco quantificationally, which is faster, more sensitive and specific.
- Real-time PCR /
- PVY /
- detection

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参考文献(6)

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