摘要
目的 为了使液相交在检测更加灵敏、特异、快速。方法 通过改进杂交液的成分 ,选择最佳的探针浓度 ,使 5’端标记生物素的PCR扩增产物与 5’端标记地高辛的探针在PCR反应管中进行液相杂交 ,杂交条件为 :94℃ 10s冷却到 37℃ 10s即完成杂交。杂交后产物通过链霉素亲和素被固定在微孔表面 ,同时在含高浓度DMSO的杂交液中将非特异结合的探针解离 ,然后对被固定的特异杂交产物进行ELISA检测。结果 液相杂交的条件优化 :杂交液中DMSO浓度为 30 0ml/L ,探针浓度为 0 5 μmol/L ,杂交温度为 37℃。对 6 0bp～ 6 0 0bp的PCR产物的杂交特异性无明显差异 ,可以检测到 1× 10 4copy的PCR产物 ,对 10 0例乙型肝炎患者的血清进行检测 ,HBsAg(+)、HBeAg(+)、抗HBc(+)的血清检出HBVDNA阳性为 10 0 % (30 / 30 ) ;HBsAg(+)、抗HBe(+)、抗HBc(+)的血清检出HBVDNA阳性为 10 0 % (30 /30 ) ;HBsAg(+)、抗HBe(+)、抗HBc(+)的血清检出HBVDNA阳性为 78 6 % (2 2 / 2 8) ;HBsAg(+)、抗HBc(+)的血清检出HBVDNA阳性为 6 6 7% (16 / 2 4 ) ,其余为阴性。结论 该液相杂交法操作简单、快速、灵敏、特异 。
Objective to make liquid phase hybridization assay more rapid, sensitive, specific.Methods Through optimizing ingredient of hybridization solution and concentration of probes, the hybridization of 5' biotin labeled products of PCR and 5' digoxin labeled probes in PCR tube can be carried out in a thermocycler for 10 seconds at 94℃, and for 10 seconds at 37℃. Then the hybridized products were immobilized on microplate wells coated with streptavidin, meanwhile the nonspecifically hybridized probes were removed in the solution of high concentration DMSO. finally, the specifically hybridized products lived on the surface of microplate wells were detected by ELISA.Results The optimized condition of liquid phase hybridization was that the temperature of hybridization was 37℃, and the concentration of DMSO and probe was 300 ml/Land 0.5μ mol/L respectively. Significant difference of specificity was not found when the different length products or PCR were hybridized, the sensitivity of hybridization is that 1×10 4 copy products of PCR can be detected. After serum samples of 100 patients with chronic hepatitis were tested, it was found that the positive rates for 30 serum samples with HBsAg(+)\, HBeAg(+)\,anti HBc(+) were 100%(30/30), 28 serum samples with HBs Ag(+)\, anti HBc(+) were 66.7%(16/24),the rest serum were negative.30 normal serum were all negative for HBV DNA.Conclusion the liquid phase hybridization procedure is simple, rapid, sensitive and specific, and fit in with clinical laboratory.
出处
《中国实验诊断学》
2002年第5期315-317,共3页
Chinese Journal of Laboratory Diagnosis
