| Autor: |
Silva AL; Biofidelity Ltd, 330 Cambridge Science Park, Cambridge, CB4 0WN, UK., Powalowska PK; Biofidelity Ltd, 330 Cambridge Science Park, Cambridge, CB4 0WN, UK., Stolarek M; Biofidelity Ltd, 330 Cambridge Science Park, Cambridge, CB4 0WN, UK., Gray ER; Biofidelity Ltd, 330 Cambridge Science Park, Cambridge, CB4 0WN, UK., Palmer RN; Biofidelity Ltd, 330 Cambridge Science Park, Cambridge, CB4 0WN, UK., Herman B; Agilent Technologies LDA UK Ltd, 5500 Lakeside, Cheadle, SK8 3GR, UK., Frayling CA; Biofidelity Ltd, 330 Cambridge Science Park, Cambridge, CB4 0WN, UK., Balmforth BW; Biofidelity Ltd, 330 Cambridge Science Park, Cambridge, CB4 0WN, UK. b.balmforth@biofidelity.com. |
| Abstrakt: |
Accurate detection of somatic variants, against a background of wild-type molecules, is essential for clinical decision making in oncology. Existing approaches, such as allele-specific real-time PCR, are typically limited to a single target gene and lack sensitivity. Alternatively, next-generation sequencing methods suffer from slow turnaround time, high costs, and are complex to implement, typically limiting them to single-site use. Here, we report a method, which we term Allele-Specific PYrophosphorolysis Reaction (ASPYRE), for high sensitivity detection of panels of somatic variants. ASPYRE has a simple workflow and is compatible with standard molecular biology reagents and real-time PCR instruments. We show that ASPYRE has single molecule sensitivity and is tolerant of DNA extracted from plasma and formalin fixed paraffin embedded (FFPE) samples. We also demonstrate two multiplex panels, including one for detection of 47 EGFR variants. ASPYRE presents an effective and accessible method that simplifies highly sensitive and multiplexed detection of somatic variants. |
