确定杂交液成分,选择探针浓度,将5’端标记生物素的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℃,5 min;55℃,5 min)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化乙锭,适用于转基因产品及其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.福建省财政厅资助项目(1401-621601);厦门市科技计划资助项目(3502Z2001109)
