应用地高辛标记的PCR-ELISA技术快速检测转基因水稻

Rapid and Sensitive Detection of Transgenic Rice Gene by Digoxigenin Labeled PCR-ELISA

应用地高辛标记的PCR-ELISA(Dig-PCR-ELISA)技术进行转基因水稻检测研究。针对转基因水稻中普遍存在的花椰菜花叶病毒(CaMV)35S 启动子(p35S)、胭脂碱合成酶(NOS)终止子(Tnos),筛选标记潮霉素磷酸转移酶基因(hpt,hpt)、β-葡萄糖苷酸酶基因(gus)、抗草丁膦除草剂基因(bar),建立Dig-PCR-ELISA检测方法;能进行半定量检测,敏感性试验表明,Dig-ELISA检测比常规电泳检测可提高敏感性达1 000倍,可检测含量达0.1％的GMO样品。全过程可在24 h内完成。

Digoxigenin labeled PCR-ELISA (Dig-PCR-ELISA) methods were developed for detection of 35S-promoter gene (p35S), NOS-terminator gene (Tnos), hygromycin B phosphotransferase gene (hpt ), Streptomyces hygroscopicus bar gene ( bar), and beta-glucuronidase gene (gus) that commonly existed in transgenic rice ( GMOs Rice). Several strains of GMOs Rice were successfully detected. The Dig-labeled PCR products were detected via ELISA by using specific probe capture hybridization. The methods were about 1 000 times more sensitive than detection of PCR products by agarose gel electrophoresis. The specificity of the PCR-ELISA were strengthened by applying high GC content probe sequences thus high hybridization temperature, as well as strict washing condition. Samples containing 0.1% GMOs products could be detected. The whole procedure could be finished within one day.