摘要
确定杂交液成分，选择探讨浓度，将5′端标记生笔素的PCR扩增产物与5′端标记地高辛的探针呈液相混合，在PCR管中按优化后的条件（92℃，5min；55℃，5min）进行液相杂交。杂交产物通过链霉亲和素被固定在微孔表面，同时在含高浓度二甲基亚砜（dimethyl sulfoxide,DMSO）的杂交液中将非特异结合的探针解离，然后对被固定的特异杂交产物进行ELISA检测。PCR－ELISA液相杂交检测方法操作简便，特异性高，避免了繁琐的杂交程序和对人体有害的溴乙锭，适用于转基因产品及春加工食品的快速检测。
Optimizing ingredient of hybridization solution and concentration of probes was conducted. 5'-biotinylated PCR product was automatically hybridized to a digoxigenin-labeled probe presented in the PCR reaction. The hybridization is performed as part of the PCR program. Streptavidin bound to the wall of tube were used to capture the biotin- digoxigenin- labeled fragments, meanwhile the non-specifically hybridized probes were removed in the solution of high concentration dimethyl sulfoxide (DMSO) . The biotin-digoxigenin hybrids were served in ELISA detection. Liquid-phase hybridization in PCR-enzyme linked immunosorbent assay (PCR-ELISA) method enabled a fast, specific and accu-rate detection of GMOs and thus appeared to be a useful tool for routine analysis of raw and processed food products. Laborious blotting procedures and hazardous ethidium bromide in gel staining could be avoided.
基金
福建省财政厅资助项目(1401-621601)
厦门市科技计划资助项目(3502Z2001109).
