PCR-EIA法检测端粒酶活性及其临床应用

目的 :探讨用PCR EIA法检测人端粒酶活性及其临床应用。方法 :利用kim法处理细胞和组织标本及扩增端粒酶产物 ,引物TS用生物素标记 ,CX用地高辛标记 ,PCR产物与包被有链亲和素的酶标板结合 ,并与酶标抗地高辛抗体反应 ,用TMB显色。结果 :PCR EIA法与TRAP 银染色法有很好的相关性 ,批内变异系数CV为 4 .138% ,在 30例各种恶性肿瘤组织中端粒酶活性检出率为 90 % ,2 8例癌旁组织中为 7.1%。结论 :该法具有较好的灵敏度与重复性 ,无同位素污染 ,较银染色法更为简便、快速 ,检测成本低 ,无需特殊仪器 ,适合于各级医院推广。

Quantitation of telomerase activity using the polymerase chain reaction- based enzyme immunoassay and its clinical application

Objective To investigate detective methodology of telomerase activity and its clinical application. Methods The telomerase activity was quantitated by polymerase chain reaction base enzyme immunoassay (PCR EIA). The TS primer was biotinylated (TS B), and CX primer was digoxigeninated (CX D).The amplicons containing TS B were combined with microtiter plate coated streptavidin, then combined with anti digoxigenin antibody labeled with POD and finally reacted to tetramethylbenzidine substrate solution. Results Telomerase activity measured by the PCR EIA method was comparable to that obtained from TRAP silver stain protocol. The CV of the PCR EIA method was 4.138%. Telomerase activity was detected in 90% of various tumer tissues. In control tissues, telomerase activity was detected only in 7.1%. Conclusion The PCR EIA method offers a rapid, quantitative, and nonisotopic assay for the determination of telomerase activity. It is simper than silver stain protocol. The detection of telomerase activity may play a significant role in the diagnosis of clinical tumors.