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利用正交设计L9(34)方法优化甘蔗黄叶病毒(SCYLV)抗原直接包被间接ELISA实验条件,统计结果表明,甘蔗蔗汁和蔗叶SCYLV粗提液包被抗原合适体积为150μL,最佳的抗体浓度SCYLV-IgG多克隆抗体(一抗)稀释1500倍,碱性磷酸酶羊抗兔IgG(二抗)稀释20000～30000倍,最佳反应时间为1～2h。应用这一优化体系,分析甘蔗不同部位的蔗茎和不同叶位的叶片SCYLV滴度的差异,结果表明,上部甘蔗蔗茎SCYLV病毒含量显著高于中下部蔗茎组织,最高可见肥厚带叶(+1叶)及其上一叶(-1叶)的嫩叶组织SCYLV病毒含量显著高于最高可见肥厚带叶以下第2叶(+3叶)和第4叶(+5叶)的老叶组织。甘蔗幼嫩组织部位SCYLV滴度较高,更适合于病毒检测。
The results of direct ELISA experiments showed that the optimal volume of coated antigen for SCYLV crude extract of sugarcane and sugarcane leaves was 150μL, which was the best The antibody concentration SCYLV-IgG polyclonal antibody (primary antibody) diluted 1500 times, alkaline phosphatase goat anti-rabbit IgG (secondary antibody) diluted 20000 ~ 30000 times, the best reaction time was 1 ~ 2h. The optimal system was used to analyze the difference of SCYLV titers in different parts of cane and different leaves. The results showed that the content of SCYLV in sugarcane of upper cane was significantly higher than that in middle and lower cane, 1 leaf) and its previous leaf (-1 leaf), the SCYLV virus content was significantly higher than that of the second leaf (+3 leaf) and the fourth leaf (+5 leaf) below the maximum visible hypertrophic belt leaf organization. The SCYLV titer in tender tissue of sugarcane is higher, which is more suitable for virus detection.