Development of Reverse Transcription Loop-Mediated Isothermal Amplification(RT-LAMP)System for Rapid Detection of Yunnan Tobacco Mosaic Virus Based on CP Gene
In order to rapidly detect tobacco mosaic virus(TMV)in Yunnan tobacco,five sets of prim-ers were designed for screening based on the conserved nucleotide sequence of the coat protein(CP)gene from Yunnan tobacco TMV isolate,the reaction temperature,time,betaine concentration,dNTPs concentra-tion,Mg2+concentration,and the ratio of internal to external primer concentrations were optimized by the sin-gle variable method,and then a reverse transcription loop mediated isothermal amplification(RT-LAMP)de-tection system for Yunnan tobacco TMV was established.The results showed that the optimal primer group was the fourth group,the optimal reaction temperature was 60℃,and the optimal final concentrations of Betaine,dNTPs and Mg2+were 0.6,0.4 and 2.0 mmol/L,respectively.The optimal concentration ratio of internal prim-er to external primer was 4∶1,and the optimal reaction time was 40 minutes.The results of optimized RT-LAMP could be visualized after stained by SYBR Green Ⅰ,which showed high specificity to TMV and the sensitivity was 10 times that of conventional RT-PCR.This study provided a convenient,efficient and reliable method for the detection of TMV in Yunnan tobacco.
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