化学发光法检测CYP1A1基因多态性

Methodology of detecting genetic polymorphism of CYP1A1 with enhanced chemiluminescence

目的 建立增强化学发光法 (enhancedchemiluminescence,ECL)检测CYP1A1基因多态性的方法。方法 实验方法同ELISA法 ,鲁米诺过氧化氢增强化学发光体系加入酶标板的每一个孔中 ,用单光子计数器检测其发光强度 ,即可鉴定样本基因型。结果 30个标本的检测结果与ELISA相同 ,其灵敏度比琼脂糖凝胶电泳高 10 0 0倍 ,比ELISA法高 10倍 ,辣根过氧化物酶标记DIG抗体的量为0 .2mU ,比ASA ELISA法少 ,1h杂交便可以判断标本的基因型 ,最佳杂交时间为 1.5～ 2h ,杂交温度为4 0～ 5 5℃。结论 该方法是一种更加灵敏和简便 ,极具发展前景的检测基因多态性的方法。

Objective To establish a method of enhanced chemiluminescence for detecting genetic polymorphism of CYP1A1. Methods According to ELISA, luminol-H 2O 2 chemiluminescence buffer was added to each microplate wells containing the hybridization conjugated compound, then the chemiluminescence intensity of the wells were detected using a single photon counter, and the genotype of sample was identified by chemiluminescence intensity. Results The detection results of ECL were the same as ASA-ELISA in 30 samples. The results showed that ECL was 10 and 1000 times more sensitive than ASA-ELISA and Agarose gel electrophoresis respectively. The dosage of HPR-labeled antibody of DIG was 0.2mU, This dosage was lower than that of ASA-ELISA. Only 1 hour of hybridization could identify genotype of sample, but 1.5-2.0 hours was much better, and the optimum hybridizing temperature was 40-55℃. Conclusion This method was more sensitive and cheaper for detecting genetic polymorphism.