建立PCR-ELISA定量检测HBVDNA的技术

Competitive polymerase chain reaction combining with enzyme-linked hybridization for quantification of HBV DNA

目的 建立一种敏感、特异和精确的乙型肝炎病毒基因PCR及其产物酶联杂交定量分析技术。方法 制备了一种与野生扩增片段等长的PCR内参照 ;设计了一对HBV基因组S区基因扩增引物 ,上游引物的 5′ 端用生物素修饰 ,可同时扩增长为 319bp的野生和内参照片段。两套捕获探针可分别与竞争PCR产物中的野生片段和突变片段杂交。在同一反应管内 ,加入已知量内参照及待测HBVDNA标本 ,进行PCR扩增 ,然后用酶联杂交法进行产物分析。根据Clementi等介绍的公式计算待测标本中的HBVDNA含量。结果 灵敏度达到 10 -3fmol/L。在该法中 ,由于有PCR过程中的特异引物及酶联杂交过程的特异探针两个环节的限制 ,因此具有很高的特异性。结论 竞争PCR及其产物酶联杂交定量法是一种相对精确、灵敏度很高和特异性较强的HBV基因全定量测定技术 ,优于外参照法及其它产物定量法。

Objective To develop a sensitive, specific and accurate method for quantitative detection of hepatitis B virus DNA. Methods A pair of primers (upstream primer was modified with biotin at its 5′ end) were designed to amplify a fragment of 319bp long from wild HBV S gene and mutant internal reference, respectively. Two sets of capture probe were designed, which can hybridize separately with wild fragment and mutant fragment within competitive PCR products. In a signle tube, known amount of internal reference and sample were added, thus the two templates can be amplified under the same condition with same efficiency. After 25 PCR cycles, the products were heated to denature the double stranded amplicon and then added to two DNA binding microdishes which had been previously coated with two kinds of capture probe. Hybridization occured between capture probe and one strand of the amplicon. Streptavidin alkaline phosphatase and its subtract were utilized to detect the hybridizing signal. The amount of HBV gene was calculated via OD values according to the formula introduced by Clementi et al. Results As low as 10 -3 fmol/L of HBV DNA fragment could be quantified. Moreover, high specificity were observed because one pair of gene specific primer as well as one amplicon specific probe were used in two step assay. Ten standard samples with known amount of HBV DNA were quantified singly blindly by this technique and the results revealed the tested value are quite near the true value. Conclusion Competitive PCR combining with enzyme linked hybridization of amplified products has been proven to be a method of choice for quantitative detection of HBV DNA with high sensitivity, specificity and reliability.