液相杂交技术在聚合酶链反应酶联免疫检测的应用  

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

作　　者：李稻[1] 程建新[1] 罗伟[1] 施蓉[1] 杭勤[1] 黄东生[1] 顾文莉[1] 陶义训[1] 

机构地区：[1]上海第二医科大学上海市医学检验重点实验室,200023

出　　处：《中华检验医学杂志》2001年第1期34-36,共3页Chinese Journal of Laboratory Medicine

基　　金：上海市高等学校科学技术发展基金资助(99B01)

摘　　要：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 ,5 5℃ 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∶10 0 0。液相杂交时间 3min ,固相杂交时间 12 0min ,但其检测结果无明显差别。检测灵敏度 1× 10 4拷贝 ml,34份HBsAg(+ ) ,HBeAg(+ ) ,抗HBc(+ )标本的阳性检出率为 97.1% ;34份HBsAg(+ ) ,抗HBe(+ ) ,抗HBc(+ )标本的阳性检出率 6 7.7% ;17份HBsAg(+ )、抗HBc(+ )阳性检出率 5 8.8% ;其他免疫检测指标组合标本为 34份 (5 .9% ) ;34份献血员标本均为阴性。结论　在PCR ELISA中使用液相杂交技术可使实验操作简便、快速 。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∶1?000. Significant difference was noted between liquid phase hybridization for 3 minutes and solid phase for 120 minutes, with a sensitivity of 1×10 4 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.

关 键 词：肝炎病毒 乙型 核酸杂交 聚合酶链反应 酶联免疫检测 

分 类 号：R446.6[医药卫生—诊断学]