微板酶联夹心杂交法定量检测人IL-18 mRNA

An enzyme-linked sandwich hybridization on microplate for quantification of hIL-18 mRNA

目的 :建立一种高灵敏度、高特异性、精确、简便的微孔板酶联夹心杂交技术 ,定量检测人IL 18mRNA。方法 :针对人IL 18mRNART PCR产物一条链的不同区域序列设计一对特异探针 ,其中一条为捕获探针 ,5’端为氨基修饰 ,可以与微量DNA结合板表面的NOS基团共价结合 ,“竖直”地包被在微孔板内 ;另一条为检测探针 ,3’端标记生物素。提取人外周血单个核细胞中总RNA ,进行RT PCR扩增IL 18mRNA ,产物热变性后加入已包被捕获探针的微孔板内进行杂交 ,再加入检测探针与已杂交的产物结合 ,最后经亲和素 辣根过氧化物酶 (SAV HRP)系统检测杂交信号。结果 :该法检测IL 18mRNART PCR产物的灵敏度明显高于琼脂糖凝胶电泳 ,重复性良好 ,且结果数据化。结论 :该方法具有操作简单、灵敏度高、特异性强等优点 ,适合于PCR扩增产物的定量检测。

Objective:To develop a simple, sensitive, specific and accurate method based on enzyme linked sandwich hybridization on microplate for the quantitive detection of human IL 18 mRNA.Methods:Two probes were designed, one is capture probe; with amino modified at 5' end, can couple with the N oxysuccinimide esters (NOS) group on the DNA binding wells through covalent attachment. In this way, oligonucleotides are immobilized on the plate surface and arise straightly to capture target amplicon. The other is a detection probe with a biotinylated 3' end.Total RNA was extracted from peripheral blood mononuclear cell and IL 18 cDNA was amplified by using RT PCR. Heat denatured products and detection probe, were then added to wells and hybridization occurs between capture probe ,target amplicon and detection probe. After the addition of Avidin Peroxidase developing system, the optical density were read at 450nm.Results:This method is more sensitive and reproductive than agarose gel eletrophoresis. The experiment results can also be digitalized.Conclusion:With its simplicity, sensitivity, and specificity results, this method is a good choice for quantitive detection of PCR products.