煙草PVY Real-Time PCR 定量檢測體系的建立及應用
Establishment and Application of Real-time PCR for Detection of Potato Virus Y in Tobacco
-
摘要: 馬鈴薯Y 病毒是近年來危害煙草生產的重要病毒之一,，嚴重影響煙草的產量與品質,。本研究利用DNAMAN 軟件對GenBank 數(shù)據(jù)庫中已登錄的馬鈴薯Y 病毒 (Potato virus Y, PVY) 全基因組序列進行序列比對,，設計引物，以煙草肌動蛋白基因為內參,，建立了煙草PVY 的實時定量檢測體系,。獲得的real-time PCR 擴增基線平整，指數(shù)擴增明顯,，斜率大,；穩(wěn)定性和重現(xiàn)性好，變異系數(shù)??；循環(huán)閾值與PCR 起始模板量對數(shù)之間存在良好的線性關系。與DAS-ELISA 相比,，該方法具有高效,、靈敏、特異性強等優(yōu)點,，為從分子生物學水平上檢測煙草中PVY 提供了新的技術手段,。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.
下載: