Research On New Virus Detection Technology Based On CRISPR/Cas12a And Cas13a

The COVID-19 pandemic and the continued emergence of other novel viruses pose a serious risk to public health and safety worldwide.Therefore,the development of simple and rapid detection methods to identify these viruses is of great importance to control the spread of viruses.At present,the etiological diagnosis of viruses mainly includes virus isolation and culture,immunological assay,and nucleic acid detection.PCR and isothermal amplification are the most sensitive nucleic acid detection methods.However,there are still some shortcomings of these two traditional assays.First,PCR-based assays require large and expensive cycling equipment,rely on trained personnel to perform complex and tedious operations,and require long turnaround times.Second,single isothermal amplification methods suffer from non-specific amplification.Recently,newly developed CRISPR/Cas diagnostic tools can be run at body temperature or even room temperature,which,together with the unique features of simple design,cost-effectiveness,and low labor intensity,are expected to replace traditional assays.In this thesis,we coupled CRISPR/Cas system with the isothermal amplification method and developed two cell phone-assisted virus detection platforms based on CRISPR/Cas system for rapid and ultra-sensitive detection of SARS-CoV-2 RNA and Ebola RNA,respectively.The main studies are as follows:1.RTF-EXPAR rapid isothermal amplification was combined with Cas12 a for SARSCoV-2 RNA detectionAn RTF-EXPAR rapid isothermal amplification combined with the Cas12 a detection platform was designed for SARS-CoV-2 RNA detection.RTF-EXPAR amplification is triggered when the target RNA is present,which in turn activates the CRISPR/Cas12 a system to rapidly shear the signaling molecule and release the fluorescent signal.This platform combines the advantages of RTF-EXPAR and CRISPR/Cas12 a,reduces the non-specific interference caused by isothermal amplification,and detects low concentrations of SARS-CoV-2 RNA within 40 min with only isothermal control.The detection limit was 3.77 a M(～2 copies/μL)when the fluorescence signal was read using an enzyme marker.The detection limit could reach the level of 4.81 a M(～3copies/μL)by analyzing the fluorescence intensity with the aid of a smartphone.The method is rapid,sensitive,accurate,and requires less equipment,providing a new research idea for the field detection of viruses.2.Cas13 a was combined with the DNA roller for the detection of Ebola RNAA Cas13a-binding DNA roller detection platform,Cas-Roller,was designed for the direct detection of Ebola RNA in unamplified samples.By optimizing the conditions,the platform was able to reach detection limits as low as 291 a M(～175 copies/μL)and the entire workflow could be completed in 40 min under a single temperature condition.At the same time,in order to simplify the equipment required for the operation,the fluorescence intensity analysis was assisted by a smartphone,which could detect 1.13 f M(～678 copies /μL)of the target viral RNA,and the sensitivity was comparable to that of instrumental detection.The downstream DNA roller reaction is independent of the target sequence,and the detection platform can be applied to the detection of any other RNA simply by redesigning the spacer region of cr RNA.This strategy is simple,rapid,and accurate,showing great application potential in virus detection.