T7 RNA聚合酶催化的荧光扩增技术检测结直肠癌循环肿瘤细胞hTERT

Detection of hTERT of circulating tumor cells in peripheral blood of patients with colorectal cancer by cellular fluorescence amplification catalyzed by T7 RNA polymerase technique

目的 T7RNA聚合酶催化的荧光扩增技术(Cellular FACTT)检测人循环肿瘤细胞中hTERT表达。方法以亲和素作为连接分子,连接生物素化的检测抗体和生物素化的DNA,加入T7RNA聚合酶进行转录扩增反应,对生成的RNA产物进行荧光检测,并同时用酶联免疫方法检测hTERT及CEA作为比对。结果 64例结直肠癌患者血清hTERT阳性率是45.3%(29/64),血清CEA阳性率是57.8%(37/64);Cellular FACTT方法检测循环肿瘤细胞的hTERT检出率为81.3%(52/64)。其检测的灵敏度与酶联免疫检测方法有显著差异P<0.01。结论 T7RNA聚合酶催化的荧光扩增技术较酶联免疫检测方法具有更高的敏感性,有可能作为一种新的检测方法用于临床的诊断。

Objective To detect hTERT of circulating tumor cells in peripheral blood of patients with colorectal cancer by cellular fluorescence amplification catalyzed by the T7 RNA polymerase technique. Methods Avidin was used to bridge biotinylated detection antibody and biotinylated DNA. T7 RNA polymerase was added to perform RNA amplification. RNA products of hTERT were quantified by adding the RNA intercalating dye RiboGreen, while comparing with the plasmatic expression of CEA by ELISA. Results In 64 patients, the positive rate of hTERT was 45.3% （29/64）, whereas the positive rate of plasmatic CEA was 57.8% （37/64）. The detection rate of circulating tumor cells by the cellular FACTT technique was 81.3% （52/64）. The detection of FACCT for hTERT was more sensitive than ELISA controls （P 〈0.01 ）. Conclusions FACCT has a higher sensitivity than ELISA for the detection of hTERT. It might be a powerful and sensitive tool for clinical diagnosis of circulating tumor cells in early stage.