摘要
目的 建立灵敏、快速、特异的 PCR- EL ISA检测结核菌 DNA的方法 .方法 用一对生物素标记的上游引物和未标记的下游引物对 TB IS6 110的一段 DNA进行快速 PCR扩增 ,使扩增产物的一条链上带有生物素 ,同时 PCR反应液中存在有地高辛的标记的检测探针 ,随着 PCR的结束 ,探针与生物素标记的扩增产物形成特异的杂交体 ,该杂交体通过链霉亲和素被固定在微孔板表面 .然后用标记有过氧化物酶的地高辛抗体与杂交体上的地高辛结合 ,漂洗后加底物显色 ,测定吸光度值 (A4 50 nm) .结果 该方法灵敏、特异、快速 ,可以检测出 10 copies的模板量 ,对临床 6 0例痰标本进行检测 ,结果有 38例为阳性 ,有 2 2例为阴性 ,检出率不低于培养法 .结论 该方法适用于临床进行快速的 TB基因检测 ,也可用于其它病原体的检测 .
AIM To develop a rapid, sensitive and specific method of PCR ELISA for detecting TB DNA. METHODS TB IS6110 DNA was amplified by bio labeled up stream primers and general down primers to combine a string of double strings DNA of PCR products with biotin. In the end of PCR, digoxigenin labeled probes added into PCR were hybridized with the bio labeled PCR products, forming the specific hybrid DNA with biotin and digoxigenin in both of their ends respectively, and the hybrid DNA was immobilized on the surface of the streptavidin coated wells. Then the peroxidase antidigoxigenin conjugate was linked with the digoxigenin of the hybrid DNA. Finally the substrate was added and the Ab values were measured. RESULTS This method was sensitive, rapid and specific. 10 copy templates could be detected. 60 patients' sputa were tested by this method with the result of 38 positive and 22 negative. The positive rate of the was not less than that of culture. CONCLUSION The PCR ELISA can be used for detecting TB DNA in sputum samples, and other pathogens.
出处
《第四军医大学学报》
北大核心
2002年第22期2086-2088,共3页
Journal of the Fourth Military Medical University