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根据烟草花叶病毒(Tobacco mosaic virus,TMV)外壳蛋白(CP)RNA的特异性序列设计TaqMan荧光探针及其引物,利用实时荧光定量RT-PCR检测食用菌蛋白抗TMV的活性。经食用菌蛋白处理后,TMV病毒汁液中TMV的RNA浓度下降了22.02%~87.93%,传统生物学测定的食用菌蛋白抗TMV的平均枯斑抑制率为10.88%～83.97%;相关性分析表明,实时荧光定量RT-PCR测定的病毒RNA浓度的下降与传统生物学方法测定的平均枯斑抑制率之间呈正相关(r=0.818 8),具有较好的一致性。利用实时荧光定量RT-PCR检测蛋白抗烟草花叶病毒活性的方法,具有特异性好、快速、简便、重复性高的特点,适合于蛋白抗烟草花叶病毒活性的痕量快速高效检测。
The TaqMan fluorescent probe and its primers were designed according to the specific sequence of tobacco mosaic virus (TMV) coat protein (CP) RNA, and the activity of edible fungus protein against TMV was detected by real-time fluorescence quantitative RT-PCR. The TMV RNA concentration in TMV virus juice decreased by 22.02% ~ 87.93% after treated with edible fungus protein, and the average inhibition rate of fungus protein anti TMV by traditional biology was 10.88% ~ 83.97%. Correlation analysis showed that , And the decrease of viral RNA concentration by real-time fluorescence quantitative RT-PCR was positively correlated with the average inhibition rate of the malignant cells determined by the traditional biological method (r = 0.818 8), which showed good consistency. The method for detecting anti-tobacco mosaic virus activity by real-time fluorescence quantitative RT-PCR has the advantages of good specificity, quickness, simplicity and high repetition. The method is suitable for rapid and efficient detection of protein anti-tobacco mosaic virus activity.