液相杂交技术在聚合酶链反应酶联免疫检测的应用

目的采用液相杂交技术，简化核酸杂交过程，建立乙肝病毒（HBV）的液相杂交的聚合酶链反应-酶联免疫检测（PCR-ELISA）方法。方法将5′端标记生物素的PCR扩增产物与5′端标记地高辛的探针呈液相混合，90℃2min，55℃1min杂交。杂交后产物被链霉亲和素酶标板固定，经酶标记抗地高辛标记抗体结合、显色。结果液相杂交酶联免疫检测条件优化探针浓度3pmol/次,酶标记抗体稀释度1∶1000。液相杂交时间3min，固相杂交时间120min，但其检测结果无明显差别。检测灵敏度1×10

Liquid-phase hybridization in polymerase chain reaction-enzyme linked immunosorbent assay

Objective　To develop a liquid phase hybridization technique for PCR-ELISA.Methods　Hepatitis B virus (HBV) DNA was used as a target DNA and its PCR products were biotin-labeled by a 5′-biotin-labeled primer through sample amplification.After the labeled product was mixed with a 5′-digoxinlabeled probe,hybridization was carried out in a thermocycler for 2 minutes at 90℃ and for 1 minute at 55℃.Then the hybridized product was captured on microplate wells coated with streptavidin and was detected with a peroxiase-labeled antibody and TMB substrate.Results　The optimial concentration of probes was 1.5 μmol/L 2 μl(3 pmol/well) and enzyme-labeled anti-digoxigenin antibody was 1∶1000.Significant difference was noted between liquid-phase hybridization for 3 minutes and solid-phase for 120 minutes,with a sensitivity of 1×104 copies/ml for virus DNA.The positive rates for 34 serum samples with HBsAg(+),HBeAg(+),anti-HBc antibody(+) were 97.1%; 34 samples with HBsAg(+),anti-HBe antibody(+) and anti-HBc antibody(+) 67.7%; 17 samples with HBsAg(+),anti-HBc antibody(+) 58.8%,and 34 samples with other HBV serological markers(+) 5.9% respectively.The specificity was 100%.Conclusion　The liquid-phase hybridization technique for PCR-ELISA,is simple,rapid and suitable in clinical laboratory.