A New One-step Nucleic Acid Real-time Detection Technology Based On The Direct-amplification ERA-coupled CRISPR/Cas12a Gene Editing System

Herein,we introduced a new approach for nucleic acid detection: One-Pot Realtime Nucleic Acid Detection by Direct ERA Combined with CRISPR/Cas12 a.Enzymatic Recombinase Amplification(ERA)relies on three core enzymes employed: Recombinase,single-stranded DNA-binding protein(SSB)and stranddisplacing polymerase.Under isothermal conditions,ERA is able to achieve in vitro nucleic acid amplification using recombinase and single-stranded binding proteins that act synergistically at room temperature to achieve specific binding of primers to the template.Therefore,this isothermal amplification technique has decreased the reliance on thermal cycling instruments and is suitable for rapid detection in primary laboratories or in the field.The advent of the CRISPR/Cas toolbox has revolutionized genome engineering fields and facilitated the preparation of gene knockout models,functional gene analysis,and specific nucleic acid sequence detection.With the study of CRISPRrelated protein Cas12 a protein,Cas proteins have been demonstrated to possess nonspecific endoribonuclease activity when the Cas-cr RNA complex is bound to its target,degrading sequences supplied either in trans or in cis with the target.Adding to CRISPR/cas12 a reaction when the probe is intact,the fluorescent signal emitted by the luminescent group is absorbed by the quenched group;however,when the Cas12 a trans-cleavage activity is activated,the ssDNA fluorescent probe is indiscriminately cleaved,so that the fluorescent emitting group and the fluorescent quenched group are separated,and thus the fluorescent signal can be received by the fluorescent monitoring system,namely,the accumulation of fluorescence signal and the synchronization of target detection are realized,thus realizing real-time detection.Using Staphylococcus aureus,barley yellow mosaic virus,and transgenic maize TC1507 samples as materials,crude sample extraction by alkaline lysis,ERA amplification of target sequence,Cas12 a protein as a highly specific sequence recognition element,we established a nucleic acid detection kit method that can be used for quick,accurate,real-time,test DNA or RNA templates,and further improved the kit process by blow-drying or lyophilizing each reaction reagent involved in the experiment,namely Pre-dispensing.The addition of alginose to maintain the enzymatic activity of the reaction reagents.In the field testing,the reagents can be opened only once to achieve the foolproof operation of "sample in-result out",which not only simplifies the field operation steps,but also effectively prevents contamination,without the need for professional laboratory and professional personnel.With the use of a portable real-time thermostatic fluorescence detector,it is more suitable for field testing and on-site testing,broadening its application scenarios.It was demonstrated that the ERA-Cas12 a method designed in this study detects Staphylococcus aureus with high specificity,no crossover with other food-borne viruses,its sensitivity to 10 copies,and sensitivity of 10 CFU/reaction for food samples contaminated with S.aureus;detection of barley yellow mosaic virus,except for barley yellow mosaic virus,all other viruses have no amplification curve,and RNA samples can be detected at concentrations above 0.001 ng/?L.Detection of transgenic maize TC1507,only transgenic maize TC1507 samples have an amplification curve,the LOD of this method is 10 copies.In summary,the ERACas12 a method included in this study has great application prospects and can provide a new idea for the detection of plant and animal viruses.