PCR-微孔板杂交检测端粒酶活性方法的建立及初步应用

目的建立聚合酶链反应-微孔板杂交法检测端粒酶活性的方法及应用于临床。方法利用Kim法处理样品及扩增端粒酶产物,引物TS标记生物素,扩增产物与包被有亲合素的微孔板结合,并与标记地高辛特异探针杂交;与碱性磷酸酶标记的抗地高辛抗体反应,经PNPP显色。结果该方法最佳实验条件为探针浓度2.5μmol/L,杂交时间1h,批内变异系数为9.5%,批间变异系数为16.5%。在29例各种恶性肿瘤组织,端粒酶活性总检出率为89.6%,而在17例炎性包块与良性增生组织为11.8%,7例正常组织中未发现有端粒酶活性。结论该法灵敏度与重复性较好,简便快速,成本低,便于临床推广。

Detection of Telomerase Activity Using PCR-microtiter Plate Hybridization

Aim　To establish a PCR-MPH method for telomerase activity and its clinical applicatiom.Methods　The tel-omerase activity was detected by PCR-MPH,which amplicons containing biotin and TS primer was conbined with microtiter plate coated avidin,then hybridized with digoxin probe and finally combined with anti-digoxin-AKP.Results　The optimum concentration of digoxin probe was 2.5 μmol/L.Hybridization time was 1 hour,hybridization temperature was 37 ℃.The within-run CV was 9.5%,the between-run CV was 16.5%.The positive rate of telomerase was 89.6% of patients with various tumour tissues,and 11.8% of patients with inflammation as well as benign proliferation tissue,while telomerase activity was not detected in 7 normal tissues.Conclusion　The assay has better sensitivity,producibility.It is simple and rapid.