A quantitative method to measure telomerase activity by bioluminescence connected with telomeric repeat amplification protocol.
A quantitative method to measure telomerase activity by bioluminescence connected with telomeric repeat amplification protocol.
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DOI：
10.1006/abio.2001.5418
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发表时间：
2001-12
影响因子：
2.9
通讯作者：
Shun-qing Xu;Minming He;Hong-ping Yu;Xiaokun Cai;Xianglin Tan;Bin Lu;Baihuai Shu
中科院分区：
文献类型：
--
作者：
Shun-qing Xu;Minming He;Hong-ping Yu;Xiaokun Cai;Xianglin Tan;Bin Lu;Baihuai Shu
端粒酶有望成为一种新的肿瘤诊断标志物。端粒重复序列扩增法（TRAP）是检测端粒酶活性的一种敏感方法。然而，TRAP及其改进的方案并不总是适合于测量大量临床样品的端粒酶活性以诊断癌症，因为这些方法通常需要耗时的检测步骤，例如凝胶电泳。为了改进质量诊断的程序，我们应用生物发光来代替检测步骤。通过使用灵敏的酶促发光无机焦磷酸盐检测分析（ELIDA）评估端粒酶延伸产物的PCR扩增中产生的无机焦磷酸盐的量来测量端粒酶活性。TRAP-ELIDA法可在2 ~ 1000细胞当量范围内定量检测端粒酶活性。ELIDA信号与TRAP-SYBR绿色染色结果一致，且ELIDA与TRAP-酶联免疫吸附试验（TRAP-ELISA）结果呈显著相关（r（2）= 0.992，P < 0.001）。TRAP-ELIDA是一种简便、灵敏的检测端粒酶活性的方法。由于TRAP-ELIDA用光度计测量端粒酶活性，因此可同时应用于大量临床样本。
Telomerase is expected to be a new biomarker for cancer diagnosis. The telomeric repeat amplification protocol (TRAP) is a sensitive method to detect telomerase activity. However, TRAP and its modified protocols are not always suitable for measuring telomerase activity of a large number of clinical samples to diagnosis cancer because these methods generally require a time-consuming detection step such as gel electrophoresis. To improve the procedure for mass diagnosis, we applied bioluminescence to replace the detection step. Telomerase activity is measured by evaluating the amount of inorganic pyrophosphate generated in PCR amplification of telomerase elongation product, with use of the sensitive enzymatic luminometric inorganic pyrophosphate detection assay (ELIDA). TRAP connected with ELIDA (TRAP-ELIDA) can quantitatively detect telomerase activity within linearity from 2 to 1000 cell equivalents. The ELIDA signals accorded with results of TRAP-SYBR green staining, and the results of ELIDA were significantly correlated to those of TRAP connected with an enzyme-linked immunosorbent assay (TRAP-ELISA) (r(2) = 0.992, P < 0.001). TRAP-ELIDA is a simple and sensitive method to quantify telomerase activity without time-consuming gel electrophoresis. Because TRAP-ELIDA measures telomerase activity with a luminometer, it could be applied to a large number of clinical samples at the same time.