Real-time detection of telomerase activity utilizing duplex scorpion primers and hairpin-like reverse primers

Telomerase, a ribonucleoprotein enzyme, is expected to be a new marker for cancer diagnosis. The development of the telomeric repeat amplification protocol (TRAP) and its modified versions have facilitated the detection of telomerase activity in small tissue and tumour samples. But most of these techniques require complex post-PCR procedures. As for the two real-time quantitative methods (SYBR Green and Amplifluor methods) reported so far, both use fluorogenic probes without specificity. To overcome these problems we developed a new real-time method for the detection of telomerase activity. In this method a duplex scorpion primer and reverse primers with hairpin-like structures were used. The use of duplex scorpion primers shows a series of advantages: the target-specific probe sequence provides higher specificity than the SYBR Green and Amplifluor methods; the unimolecular probing mechanism allow the assay to be conducted under fast cycling conditions and a single operation can be completed in 1.5 h; the closed-tube system reduces the risk of carryover contamination and supports high throughput. This method may be a useful tool to rapidly, specifically and precisely quantify telomerase activity.