Rapid and Sensitive Diagnosis of Drug-Resistant FLT3-F691L Mutation by CRISPR Detection

Abstract

Sensitive and efficient detection of drug-resistant mutations is essential in cancer precision medicine. In treating acute myeloid leukemia (AML), FLT3 gene F691L mutation shows universal resistance to all currently available FLT3 inhibitors. However, there is no particular detection method for FLT3-F691L. Commonly-used first-generation sequencing (FGS) approaches have low sensitivity, and next-generation sequencing (NGS) is time-consuming. Herein, we developed an accurate and sensitive FLT3-F691L diagnostic method by CRISPR detection. Briefly, the FLT3-691 region is amplified by recombinase polymerase amplification (RPA) and detected by L691-crRNA induced Cas12a reaction, and finally the result can be directly observed under a blue lamp or analyzed by a fluorescence reader. Confirmed by the tests on diluted plasmids and 120 AML patient samples, this method can achieve a sensitivity of 0.1% and complete the whole diagnosis process within 40 min. Potentially, this method will play an important role in point-of-care applications and guidance of AML treatment.

Label-free DNA quantification using isothermal amplification on an exposed core optical fiber microfluidic platform

“…Suzhou Xianda Gene has developed an enzymatic recombination isothermal amplification (ERA) method, which requires only 15 min of amplification reaction time at the optimum temperature of 37-40 °C. 15,16 Its operation at low temperatures and high sensitivity were a major breakthrough, and the avoidance of complex sample processing makes it suitable for field testing where nucleic acid cannot be extracted. 17,18 The isothermal amplification method is of great significance to the fields where DNA amplification is widely used, such as genetics, 19,20 medical diagnostics, 21,22 and food safety.…”