Highly Sensitive Telomerase Assay Insusceptible to Telomerase and Polymerase Chain Reaction Inhibitors for Cervical Cancer Screening Using Scraped Cells

5 years ago

Highly Sensitive Telomerase Assay Insusceptible to Telomerase and Polymerase Chain Reaction Inhibitors for Cervical Cancer Screening Using Scraped Cells

Daisuke Miyoshi, Hidehiko Okazawa, Yasushi Kiyono, Yoshio Yoshida, Hidenobu Yaku, Yuko Fujita

A sensitive telomerase assay based on asymmetric-polymerase chain reaction (A-PCR) on magnetic beads and subsequent application of cycling probe technology, STAMC, which is insusceptible to DNase and PCR inhibitors, was for the first time applied to clinical specimens in addition to a conventional telomeric repetitive amplification protocol (TRAP). The electrophoresis results showed that an increase in scraped cervical cancer cells not only reduced TRAP products but also increased smaller products, suggesting the unreliability of TRAP for clinical samples. To achieve the required sensitivity of STAMC for clinical application, the sequence and concentration conditions were explored for the forward and reverse primers for A-PCR, which resulted in a detection limit of only two HeLa cells with 1 μM TS primer (5′-AATCCGTCGAGCAGAGTT-3′) and 0.04 μM ACX primer (5′-GCGCGGCTTACCCTTACCCTTACCCTAACC-3′). Under the same primer conditions, the fluorescence signal of STAMC increased as scraped cervical cancer cells increased despite showing a negligible intensity for benign tumors. Furthermore, STAMC showed no signal for a cervical cancer patient treated with irradiation therapy. These results indicate that STAMC is useful for not only cervical cancer screening but also investigating the effect of cancer treatments such as radiation therapy and drug administration.

Publisher URL: http://dx.doi.org/10.1021/acs.analchem.6b04777

DOI: 10.1021/acs.analchem.6b04777