核酸杂交的酶放大时间分辨荧光分析  

Enzyme -amplified Time -resolved Fluorescence Detection for Nucleic Acid Hybridization Assays

作　　者：赵启仁[1] 李美佳[1] 刘洁[1] 宋娜玲[1] 陈艾[1] 庄湘莲[1] 刘炳辰[1] 

机构地区：[1]中国医学科学院中国协和医科大学放射医学研究所实验核医学研究室,天津300192

出　　处：《中国医学科学院学报》2002年第1期84-88,共5页Acta Academiae Medicinae Sinicae

基　　金：国家自然科学基金(39770216)~~

摘　　要：目的为了建立一种新的非同位素的核酸杂交的测定方法－酶放大时间分辨荧光分析法（ＥＡＴＲＦＡ）或酶放大镧系元素发光分析法（ＥＡＬＬ），使核酸杂交分析能更广泛地应用于临床诊断。方法将生物素－链霉亲和素的高亲和力和放大作用、酶放大作用、镧系元素螯合物的固有优点和时间分辨测量技术结合起来。通过碱性磷酸酶把５－氟水杨酸磷酸酯转变为５－氟水杨酸（５－ＦＳＡ），５－ＦＳＡ与Ｔｂ３＋和ＥＤＴＡ形成发荧光的三元复合物。结果对靶ＰＢＲ３２２ＤＮＡ测定的标准曲线范围宽，可达三个数量级；灵敏度为１０ｐｇ；准确度为９０％～１１５％。研究和优化了紫外灯照射时间，生物素化探针、ＡＰ－ＳＡ、５－ＦＳＡＰ和Ｔｂ－ＥＤＴＡ等的浓度以及洗涤方法等主要影响因素。发现了新的荧光稳定技术。结论核酸杂交的ＥＡＴＲＦＡ或ＥＡＬＬ是一种灵敏的无需转移的简单的全新分析方法，有很好的应用前景。Objective To develop a new nonisotopic detection method of enzyme -amplified time -resolved fluorescence(EATRF)or enzyme -amplified lanthanide luminescence(EALL)for nucleic acid hybrid-ization assays,which can be applied extensively in clinical diagnosis.Methods The method combines the high affinity of biotin-streptavidin system,amplification of enzyme,and inherent advantage of lant -hanide chelate with the background elimination of time -resolved fluorescence detection.The conversion of5-fluorosalicyl phosphate to5-fluorosalicylic acid(5-FSA)by alkaline phosphatase.The salicylic acid product forms a luminescent ternary chelate with Tb3+ and EDTA.Results The dynamic range of the standard curve of EATRFA for nucleic acid hybridization assay was very wide,the range was more than third order of magnitude.The detection sensitivity was about 10pg of target sequence.When the known target sequence was20,10and2ng,the ratio of measured amount to known am-ount was110%,90%and115%respectively.The main experimental conditions,for example,the irradiating time of ultraviolet rays,the concentrations of biotinylated probe,AP-SA,5-FSAP and Tb-EDTA and the methods of washing in the related steps,have been optimized.A new stable technology of fluorescence has been developted.Conclusions EATRF detection for nucleic acid hybridization assays is a new sensitive simple method,which has a great prospect.

关 键 词：核酸杂交技术 酶放大时间分辨荧光分析 酶放大镧系元素发光分析 

分 类 号：Q503[生物学—生物化学]