Highly Sensitive Detection Of Environmental Microcystins And Novel Coronavirus Based On CRISPR-Cas12

The use of simple detection methods to monitor trace pollutants and viruses in environmental water is essential for environmental safety and human health.For example,with the occurrence of climate change and eutrophication of water bodies,cyanobacteria proliferate globally,and more and more drinking water sources are polluted by microcystis toxins released by cyanobacteria,posing a great threat to human health.And infection with severe acute respiratory syndrome coronavirus 2(SARS-CoV-2)may cause the coronavirus pneumonia 2019(COVID-19),causing serious harm to the normal life of people around the world.At present,the main detection of these pollutants and viruses still relies on precision instruments.High performance liquid chromatography(HPLC)and liquid chromatography-mass spectrometry(LC-MS)are the commonly used detection methods for microcystins.The common detection methods for SARS-CoV-2 are reverse transcription polymerase chain reaction(PT-PCR).Their complex preprocessing procedures and requirements for heavy and expensive equipment limit the use of these methods for rapid and field detection.In addition,the requirement for trained technicians and ultra-clean working environments is a challenge for large-scale detection.CRISPR-Cas12a is an RNA-directed,DNA-targeted Class II CRISPR-Cas system that can be programmed with crRNA to cut specific DNA targets.Therefore,the CRISPR-Cas system can perform both recognition and signal transduction tasks at the same time,which is highly valuable for biosensing detection.In this paper,based on CRISPR-Cas 12a system combined with the corresponding signal amplification and reading system,two new biosensors are constructed and used for environmental detection.Project 1:Monitoring trace organic pollutants in situ by simple and convenient methods is very important for environmental pollution control.Here,we propose an aptamer sensor platform based on CRISPR-Cas12a for the field and sensitive detection of microcystin LR(MC-LR).After hybridization with blocker DNA,MC-LR aptamer was coupled with magnetic bead to obtain MB aptamer sensor.In the presence of MC-LR,their interaction with aptamers is triggered and specific binding results in the release of the blocker DNA.Utilizing the programmability of the CRISPR-Cas system,the released blocker DNA is designed to activate the Cas12a-crRNA complex.Single-stranded DNA reporter genes are rapidly cleaved by complexes.Signal readout can be achieved by fluorimeter or lateral flow strips,which is positively correlated with MC-LR concentration and detection limits are 3.02×10-6 μg/L(fluorescence method)or 1×10-3 μg/L(lateral flow assay).In addition,the aptamer sensing platform showed good selectivity and recovery,proving their good applicability to actual water sample analysis.During the entire test process,only two steps of incubation were required at constant temperature,and the results were visually visible when employing flow strips.The proposed detection method provides a simple and convenient alternative for in-situ MC-LR monitoring,which may have great prospects for future environmental monitoring.Project 2:At present,there is no standard detection method for novel coronavirus in sewage in China.Although different laboratories use the same method,their operations are not uniform,resulting in different test results.Therefore,an approach that is simple and accurate enough to provide reliable results for targeted prevention and control is urgently needed.Here,using G-quadruplex DNAzyme as signal reader,we established a colorimetric method for detecting SARS-CoV-2 virus in the environment by using reverse transcription-recombination polymerase amplification technique(RT-RPA)combined with clustered regularly interspaced short palindromic repeats and associated protein 12a(CRISPR-Cas12a).First,a strong base is used to break the virus directly,releasing the target nucleic acid.Then,the N-region sequence of viral RNA was amplified by RT-RPA.The amplified product can specifically activate Cas12a-crRNA,thus cleaving G4 chain(single-stranded DNA)and preventing it from forming G-quadruplex,thus failing to exhibit peroxidase activity and failing to catalyze the oxidation of ABTS2-by hydrogen peroxide.The specific manifestation was color change,which could be directly observed by naked eyes.Detection was limited to 1 copy/μL and showed good linearity between 1 and 20 copies/μL.In addition,the detection methods showed good selectivity and recovery rate,which proved their good applicability to the actual environment detection.This project also optimizes the key substances in the cache solution so that the amplification of RT-RPA is combined with the cleavage of Casl2a-crRNA and the formation of G-quadruplex DNAzyme in a test tube.At the same time,the test time is shortened,and the pollution which may be caused by switching the tube cover is avoided.We successfully constructed a biosensor platform based on CRISPR-Cas12a system for the detection of microcystin-LR and SARS-CoV-2 in the environment respectively.Both of the two detection methods showed high sensitivity and strong specificity,which is simple and accurate and has good applicability to the actual environmental detection,and is expected to be developed into environmental detection kits.