The Novel Method For Ultrasensitive Detection Of Leukemia Fusion Gene Based On DNA Machine And Nucleic Acid Isothermal Amplification Technology

The BCR-ABL1 fusion gene is considered to be the most important molecular marker of chronic myelogenous leukemia(CML),and its expression level has indispensable clinical significance for early diagnosis,efficacy monitoring,and prognosis judgment.At present,the detection methods of BCR-ABL1 fusion gene mainly conclude real-time PCR,fluorescence in situ hybridization(FISH),flow cytometer and so on.However,some defects in these methods cannot be ignored,such as complicated operation,long detection time and difficulty in point-of-care testing.Therefore,it is still urgent to develop a novel,sensitive,rapid and simple biosensing strategy for BCR-ABL1 fusion gene detection.Herein,a dual signal amplification fluorescence biosensing strategy has been developed for ultrasensitive detection of the BCR–ABL1 fusion gene based on target-triggered DNA nanomachine and multi primer-like rolling circle amplification(RCA).In this detection strategy,dual single-stranded DNA probes are utilized to specifically recognize the BCR-ABL1 fusion gene and hybrid with each other to form a T type DNA nanostructure.Then,on the basis of target-induced nanostructure,DNA machine operate efficiently under the drive of polymerase and nicking endonuclease,producing a large number of nicking products.These products serve as the primer of downstream RCA reaction,and the circle DNA template is well-designed with two nicking endonuclease recognition sites,which can lead the RCA reaction to a multi-primer amplification model.The final production,G4 nanozyme,can catalyze ThT to obtain a strong fluorescence signal.The efficient combination of dual signal amplification methods achieves the ultrasensitive detection of BCR-ABL1 fusion gene with a limit of detection as low as 5.52 fM,and generates a wide dynamic linear range from 10 fM to 1 nM,which is superior to previous similar nucleic acid detection strategies.In addition,the dual amplification reaction requires only one sample operation to complete whole detection within 60 min.Moreover,the dual-probe recognition mode effectively avoids the interference of BCR gene,ABL gene and others,so that this method can also achieve specific detection of spiked samples in a complex matrix environment.Therefore,this work has established an ultrasensitive,specific,efficient and simple BCR-ABL1 fusion detection strategy,which will provide a strong support for clinical diagnostic of CML.