不对称PCR杂交显色方法的建立及其对HBV DNA的检测

Hybridization colouring method of detecting the products of HBV DNA ASY PCR

目的：建立一种灵敏、快速、特异的检测 Ｈ Ｂ Ｖ Ｄ Ｎ Ａ Ｐ Ｃ Ｒ产物的杂交显色方法． 方法：使用一个生物素标记的上游引物对 Ｈ Ｂ Ｖ Ｄ Ｎ Ａ 进行不对称 Ｐ Ｃ Ｒ扩增，使扩增出的单链带有生物素，并可结合在固定有链亲合素的微孔板表面． 用标有辣根过氧化物酶的探针与固定的单链 Ｐ Ｃ Ｒ 产物杂交，而后加底物显色测定吸光值． 结果：该方法比常规的琼脂糖凝胶电泳法更特异，而且灵敏度高１０ 倍～１００ 倍，对简便处理的标本适应性强，具有良好的重复性． 结论：该方法可以代替琼脂糖凝胶电泳法，有望成为 Ｐ Ｃ Ｒ 产物定性检测的常规临床检验方法．

AIM: To establish a simple rapid and sensitive hybridization method detecting products of asymmetric PCR(asy PCR) for HBV DNA from serum.METHODS:The upstream primer of Asy PCR of HBV DNA was modified with biotin and it′s concentration is 50 times of that of the downstream primer in the PCR system, so that the most products of PCR were single strand and labeled with biotin. Then the products were fixed upon the streptavidin coated wells. HRP labeled probes were added into wells ; Hybridization was completed in a short time and the latest hybridization signal was detected by ELISA liked assay. RESULTS: the sensitivity of the method was 10 100 times higher than that of the electrophoresis ; to the template purified with simple method, it`s adaptability and repeatability was better. CONCLUSION: The method is of potential use for routine examination of clinical specimens.