用聚合酶链反应微孔板杂交技术检测HBV DNA

Detection of Hepatitis B Virus DNA in Serum by Polymerase Chain Reaction and Microtiter Plate Hybridization

目的 :建立并优化HBVDNA的PCR 微孔板杂交技术检测体系 ,使临床PCR结果判定更加客观可靠。方法 :采用碱裂解标本中的HBV、玻璃粉吸附提纯模板DNA ,用 5’端标记生物素的引物进行PCR ,PCR扩增产物与包被于微孔板中的靶基因杂交 ,酶标链霉亲和素与杂交体中的生物素结合后 ,用酶底物与所标记酶进行显色反应 ,最后通过测定其光密度来判定结果。结果 :建立并优化的PCR 微孔板杂交检测方法提高了检测的灵敏度和特异性。PCR 微孔板杂交法对HBVDNA的检出阳性率 (79.8% )比电泳法 (6 9.0 % )高 ,2 0份非乙肝患者血清标本两方法检测均阴性。结论 :本方法灵敏、特异、操作简便、快速、结果客观可靠。

Objective: To develop and optimize a microtiter plate hybridization assay for the detection of PCR amplicons of HBV DNA from serum. Methods: HBV virions in serum were lysaed by NaI,and DNA template purified with silicon-absorption was amplified by 5'end biotin-labelled primers. The amplicons were detected by hybridization with capturing sequences precoated onto microtiter wells, and the hybrids were visualized by colorimetry. Results: The method developed by us could detect HBV DNA with high sensitivity and specificity. This method had higher positive rate(78.9%)than PCR-electrophoresis(69.0%). As to the 20 HBsAg-negative samples,both methods,hybridization and electrophoresis, got negative results. Conclusion: This rapid method can objectively detect HBV DNA from serum with high sensitivity and specificity.