Research On Visual Virus Detection Based On CRISPR/Cas Technology

Viral infections and deadly viral diseases have seriously affected human health.Viruses can infect almost any living organism with a cellular structure.They can cause a variety of diseases and have serious consequences on human health and social stability.Due to the urgency and severity of viral infections,accurate,reliable,fast,easy-to-use,and low-cost detection methods are necessary.Polymerase Chain Reaction（PCR）is the gold standard for virus detection in clinical practice,but it has high requirements for experimental environment and operators,and the need for precision thermal cycling apparatus,which greatly affects the efficiency of virus detection.Clustered regularly interspaced short palindromic repeats（CRISPR）have made great achievements in the field of life science since their discovery.CRISPR-effecting proteins,represented by Cas12 and Cas13,have provided new directions in the field of virus detection due to their unique trans-cleavage activities.However,the current application also faces new challenges.Most of the virus detection methods based on CRISPR technology require large equipment,such as the requirement of fluorescence quantitative instruments for traditional fluorescence-based CRISPR detection;the requirement of electrical signals instruments for electrochemical sensor-based CRISPR detection;the requirement of expensive and sophisticated instruments for raman scattering spectral sensor-based CRISPR detection,which limits the application of CRISPR technology in virus detection.In view of the current status and development trend of CRISPR technology,this study uses CRISPR/Cas technology as the core,in conjunction with isothermal amplification technology and magnetic bead-based cascade amplification reaction,while utilizing gold nanoparticles（AuNPs）and lateral flow strips.A series of visual virus detection methods that were easier to read,were constructed to replace the traditional fluorescent signal.The first is the construction of a visual viral nucleic acid detection based on CRISPR/Cas12a（CLAP detection assay）.Severe Acute Respiratory Syndrome Coronavirus 2,SARS-CoV-2)was the object of detection.In this study,Reverse transcription-loop-mediated isothermal amplification（RT-LAMP）,CRISPR/Cas12a detection and functionalized AuNPs probe sensor were integrated.The CRISPR/Cas12a system recognizes the target sequence and converts the diagnostic results of the target nucleic acid into color changes under the function of the functionalized AuNPs probe sensor.At the same time,in order to further improve the sensitivity of the reaction,RT-LAMP was added before CRISPR detection in this study to achieve exponential amplification of the nucleic acid target.From RT-LAMP to CRISPR detection and the final visual signal output,the entire process is constant temperature,without the need for sophisticated professional thermal cyclers.The results showed that under optimal conditions,the assay could detect 4 copies/μL of viral RNA within 40 min.At the same time,the RNA which was extracted from cells transfected by plasmid was detected to investigated the clinical feasibility.In addition,the whole reaction can be realized by relying on only one metal bath,which reflects the POCT feasibility of this detection method.Next,a detection assay for visual viral nucleic acids based on CRISPR/Cas12b（SCAN assay）was developed.The detection platform integrated RT-LAMP amplification,CRISPR/Cas12b enzyme detection and AuNPs probe based visual detection platform,which could detect SARS-CoV-2 RNA on visual platform and fluorescence platform.Due to the heat-resistant activity of Cas12b,RT-LAMP and Cas12b detection can be integrated into one tube,briefly,RNA is recognized by cr RNA after reverse transcription isothermal amplification into DNA,and the trans-cleavage activity of Cas12b is activated,without any additional operations in the whole process.First,the buffer and reaction temperature of the integrated reaction of RT-LAMP and CRISPR/Cas12b were optimized.The results showed that the reaction conditions of 50m M KCl,10 m M Mg Cl₂ and 60℃had the best performance.Then,the feasibility of the reaction was verified step by step,and the detection limit was 4 copies/μL of viral nucleic acid.The feasibility of the integrated one-tube reaction was then verified,showing that 400 copies/μL of viral RNA could be differentiated from the blank control group at 90 min.Because the RT-LAMP and CRISPR/Cas detection are integrated in one tube,the operation process is reduced and the problem of nucleic acid contamination caused by multiple operations is avoided.Then there is the construction of a label-free visualization of viral nucleic acid detection platform based on CRISPR/Cas12a（LFP assay）,which integrates RT-LAMP,CRISPR/Cas12a system and a label-free AuNPs colorimetric system for the detection of SARS-CoV-2 RNA.The feasibility of unlabeled AuNPs colorimetric system was first verified,and the results showed that ss DNA could protect AuNPs from aggregation in salt solution,while ds DNA could not.Then,the unlabeled colorimetric system was optimized to further determine the salt concentration and ds DNA concentration in the reaction,the salt concentration was finally determined to be 100 m M and the DNA concentration is 400 n M.The feasibility and sensitivity of the reaction were verified.The detection limit was 4 copies/μL of viral RNA.With this approach,the target RNA can be identified by a smartphone-based detector or by the naked eye,making the operation process faster and more convenient for POCT in resource-limited areas.Because AuNPs is label-free,this detection method will be more cost-effective than traditional complex labeling or surface functionalization chemical methods.Finally,a visualization platform for simultaneous detection of nucleic acid and proteins based on CRISPR/Cas double enzyme digestion system was established（MCD assay）.The platform integrates magnetic separation technology,nucleic acid hybridization technology,antigen and antibody binding technology,CRISPR/Cas12a system,CRISPR/Cas13a system and lateral flow test strips,and finally realizes the visual output of results.The magnetic separation captures the target molecule,multiple target molecules can be combined on a magnetic bead,and one target molecule can be combined with an AuNPs probe.Multiple detection DNA/RNA on an AuNPs probe can be used to achieve cascade amplification of signals.The whole process is carried out at room temperature,and DNA/RNA can be identified and detected by CRISPR/Cas system to improve the specificity of the reaction.And the detection signal is finally converted into a visible test strip by lateral flow.First,viral nucleic acid and protein were detected separately.The results showed that the detection limit of viral nucleic acid was 1000 copies/μL,and the detection limit of viral protein was 100 fg/m L.Next,nucleic acid detection and protein detection were integrated into one tube to verify the sensitivity and specificity of the reaction,the detection limit of MCD is 1000 copies/μL nucleic acid and 10 pg/m L protein.In summary,four visual methods for virus detection were developed in this study.These four methods not only have high sensitivity,but also can get rid of the dependence on professional operators and professional equipment,and have a good application prospect.