乙型肝炎病毒的液相杂交技术在聚合酶链反应酶联免疫检测的应用探讨

目的 利用液相杂交技术,将核酸杂交技术进行简化,建立乙型肝炎病毒(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 以HBV为目的靶DNA,引物P15'端标记Bio-PCR扩增37个循环.PCR扩增产物与5’端标记地高辛的探针呈液相混合,85cC 2.5 min,60C1 min进行杂交,杂交后产物均经过链霉素亲和素酶标板进行固定,经过酶标仪记录抗地高辛标记抗体,结合、显色.结果 液相杂交酶联免疫检测条件的优化:地高辛的探针浓度为2.4 pmol/次,液相杂交时间为2.5min,固相杂交时间为100 min,检测结果差异无统计学意义(P＞0.05)；本试剂对87份“HBsAg+、HbeAg+、抗HBc+”标本的阳性检出率为91.95％; 72例“HBsAg+、抗-Hbe+、抗HBc+”标本的阳性检出率为68.06％,其他免疫检测指标组合标本为16份.结论 HBV的液相杂交技术应用于聚合酶链反应酶联免疫检测(PCR-ELISA),该杂交技术的实验操作简便,快速,适合于临床实验室的检测.

The exploration on application of liquid hybridization of hepatitis B virus in polymerase chain reaction enzyme-linked immunoassay

OBJECTIVE To take the use of liquid hybridization,to simplify nucleic acid hybridization techniques and establish the method of liquid hybridization of hepatitis B virus(HBV)polymerase chain reaction enzyme-linked immunoassay(PCR-ELISA).METHODS Took HBV for the purpose of target DNA,primers of P15 side marked the Bio-PCR amplification of 37 cycles.PCR products of the 5’ end tag digoxin probe was liquid mixture,85℃ 2.5min,60℃ 1min were taken for hybridization.The hybridization products were fixed by streptomycin and prime microtiter plates,anti-digoxigenin-labeled antibody was recoded,combined and colored by microplate.RESULTS The optimization of the liquid hybridization ELISA conditions:probe concentration of digoxin/time was 2.4pmol,liquid hybridization time was 2.5min,and solid-phase hybridization time was 100min,the test results had no significant differences(P﹥0.05).The detection rate of reagent for 87 "of HBsAg +,HBeAg +,anti-HBc +" specimens positive were 91.95%.The positive detection rate for 72 cases "of HBsAg +,anti-HBe +,anti-HBc +" specimens was 68.06%.The combination of detection index for other immune specimen was 16 cases.CONCLUSION The HBV liquid hybridization technology is used in the polymerase chain reaction enzyme-linked immunoassay(PCR-ELISA).The experimental operation of the hybrid technology is simple,fast,and it is suitable for clinical laboratory testing.