Novel Methods For Isothermal Amplification And Detection Of EGFR Gene Mutation Related To Tyrosine Kinase Inhibitors

With the continuous development of life sciences,the role of patients’genetic information in clinical personalized medicine has become increasingly prominent.Gene mutation detection technology is widely used in the detection of gene markers related to personalized medicine,especially in screening medication-related gene polymorphisms,guiding the use of tumor-targeted drugs,and monitoring the generation of drug-resistant mutation genes.However,with the increase in clinical demand,the problems of the existing gene mutation detection methods have gradually emerged,mainly including low detection sensitivity,high technical difficulty,high detection cost,long detection time,and contamination of amplicons.In response to the current problems,this research used the L858R site(drug sensitivity mutation)of exon 21 and the T790M site(drug resistance mutation)of exon20 of the EGFR gene related to tyrosine kinase inhibitors(TKIs)as the detection objects.We established two gene mutation detection methods based on RPA isothermal amplification technology,to provide new idea for clinical gene mutation detection with high sensitivity,high specificity,rapidity,simplicity and versatility.The methods were optimized and improved in order to establish new technologies for gene mutation detection that are more suitable for clinical personalized medicine.Firstly,based on the research of the high-sensitive RPA amplification reaction and the high-specific nucleic acid invasive reaction,we established a novel method for isothermal detection of gene mutations based on the RPA-Invader reaction.The optimal reaction conditions were determined by designing and screening RPA primers,optimizing the downstream detection probe and FEN1 enzyme required for the Invader reaction,the enzyme mixture 20×Core Reaction Mix and reaction time required for the RPA reaction.In this method,the RPA reaction was carried out at 37℃for 20 min,and then the temperature was raised to 63℃to trigger the Invader reaction,detection results can be obtained within 10 min.The detection sensitivity is up to 10~0copies/reaction,and the specificity is as high as 0.02%.The L858R mutation site can even detect mixed templates with mutations as low as 0.01%.It shows that this method can achieve high sensitivity,high specificity,high accuracy and no contamination with closed-tube detection of gene mutations in a short time,which can meet the needs of clinical detection.Use the established RPA-Invader method to detect genomic DNA extracted from cancer tissue samples and cf DNA extracted from blood samples in patients with non-small cell lung cancer,and compared the results with clinical detection results to verify its clinical value.By redesigning and screening RPA primers used to amplify short fragments,optimizing RPA primers’sequences and amplification positions,proving that the amplification sensitivity of amplifying long fragments’primers was higher.At the same time,we proposed for the first time that primers for amplifying short fragments can still be used in RPA amplification reactions.By coupling the Invader reaction that recognizes specific structures,the sensitivity can reach 10~1 copies/reaction.Secondly,considering that RPA-Invader method still requires the use of the professional fluorescence detection equipment,rapid on-site detection of gene mutations cannot be achieved in the grassroots or in resource-limited areas.Therefore,we proposed a method to combine the RPA reaction with the Invader-Au NP hybridization reaction.Through the Au NP-probe hybridization technology,the detection of the target was converted into the color change of the Au NP solution,and the result was interpreted by the naked eyes.The visual detection of gene mutations can be achieved within 90 min of the three-step isothermal amplification reaction.The detection sensitivity of this method for the plasmid templates at L858R site and T790M site were 10~0 copies/reaction and 10~1 copies/reaction,and the specificity were 0.1%,respectively,and no non-specific background signal was generated.A large number of actual clinical samples were verified for the established detection methods.Whether it is genomic DNA extracted from clinical cancer tissue samples or cf DNA extracted from blood samples,the consistency between the detection results of these two methods and the clinical detection results were more than 95%,proving the feasibility and accuracy of these novel methods.In summary,the results of this research provide a new idea for establishing rapid,simple,low-cost,high-sensitivity,high-specificity,and universal isothermal amplification detection methods for gene mutations that does not require complicated equipment.The detection of clinical samples showed that these methods established in this research can be well applied to the detection of gene mutations related to clinical personalized medicine,and has great clinical application value.The fluorescence method or visual detection can be selected according to the needs of the actual places.