用地高辛标记的PCR-ELISA技术快速检测转基因鱼

Rapid detection of transgenic fish with digoxigenin-labelled polymerase chain reaction combined with ELISA technique

取含有小鼠重金属螯合蛋白 (mMT)基因启动子及人生长激素 (hGH)基因的鱼样品 ,以Qiagene核酸纯化技术对鱼组织DNA进行纯化。常规PCR检测显示 ,mMT启动基因和hGH基因的PCR产物分别为2 4 0bp和 130bp ,与设计相符。PCR产物的核酸测序结果证实其扩增产物具特异性。地高辛 PCR(Dig PCR)标记反应显示 ,2个基因均产生 1条Dig标记的特异产物带。Dig PCR ELISA检测敏感性试验显示 ,对mMT启动子和hGH基因的检测敏感性可达 10 - 3,与常规PCR结合琼脂糖凝胶电泳检测方法相比 ,检测敏感性可提高至 10 0～ 10 0 0倍。试验证明 ,针对mMT和hGH基因所建立的Dig PCR

The fishes, with mouse metallothion (mMT) promoter gene and human growth hormone (hGH) gene, respectively, were collected from Institute of Hydrobiology (CAS), and the DNA was purified from the fish tissues using Qiagene nucleic acid purification technique. The routine PCR detection shows that the PCR products with 240 bp and 130 bp can be got respectively from mMT promoter gene and hGH gene, which corresponds with the designing. The results of nucleic acid sequence detection for PCR products verify that the amplified products have specificity. The reaction of Dig PCR shows each of the two genes can produce a band showing special product labelled with Dig. The sensitive test of Dig PCR ELISA shows that the test sensitivity of mMT promoter gene and hGH gene can get to 10 -3 , 100～1 000 times as much as the routine PCR method combined with agarose gel electrophoretic analysis. It comes to the conclusion that the Dig PCR ELISA technique for mMT and hGH genes has specific reliability.