One-step and highly sensitive quantification of fusion genes with isothermal amplification initiated by a fusion-site anchored stem-loop primer

Abstract

As causative oncogenes and drug targets, fusion genes play critical roles in tumorigenesis, development, and treatment and thus are regarded as tumor-specific molecular biomarkers. Specific identification and sensitive quantification of fusion genes are of great significance in cancer diagnosis, classification, and prognosis as well as minimal residual disease (MRD) monitoring. Herein, we proposed a specific and sensitive method for the quantitative detection of fusion transcripts by designing stem-loop primers to directly track fusion junctions of fusion genes and subsequently initiate reverse transcript loop-mediated isothermal amplification (LAMP). Benefitting from the specific and direct mechanism of stem-loop primers and the high amplification efficiency of LAMP, the proposed method can sensitively measure fusion gene transcripts with a detection limit of 100 aM (1 zmol) and achieve a wide linear dynamic range spanning at least six orders of magnitude (100 aM–100 pM). Significantly, the whole fusion transcript assay can be accomplished in one step under isothermal conditions, greatly simplifying the operation and detection processes. Meanwhile, the one-step analysis method in one tube may effectively eliminate false-positive results from product cross-contamination during multiple experimental operations and cover-opening measurements. We have demonstrated that the proposed method is practical and accurate for the quantitation of fusion transcripts in biological samples. Owing to the outstanding features of high sensitivity, excellent specificity, and simple operation, the new strategy may provide a robust and attractive platform for the quantification of fusion genes.