抗原直接包被间接ELISA检测甘蔗黄叶病毒

Detection of Sugarcane Yellow leaf virus by Direct Antigen Ccoated Enzyme-linked Immunosorbent Assay

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 L9（34） orthogonal diagram was applied to optimize detection conditions of direct antigen coated enzyme-linked immunosorbent assay （DAC-ELISA） for Sugarcane yellow leaf virus （SCYLV） in sugarcane.Statistic analyses indicated that 150 μL of SCYLV in juice and leaf crude extract antigens was the optimal volume based on 1：1 500 dilution of SCYLV-IgG and 1：20 000 or 1：30 000 dilution of alkaline phosphatase labelled goat anti-rabbit IgG.The reaction time was recommended for 1 hr or 2 hrs for the substrate coloring.Different SCYLV titer in different parts of cane and leaf were investigated using the optimized DAC-ELISA system.The titer of SCYLV was significantly higher in the upper internodes than in medium and lower internodes.In addition,SCYLV titer was significantly higher in the top-visible dewlap （TVD） leaf （＋1 leaf） and the younger leaf （-1 leaf） than that in the older leaf （＋3 leaf and ＋5 leaf）.It is concluded that younger tissue （both internodes and leaves） was better samples for SCYLV detection due to a higher viral titer