| Infectious diseases caused by viruses seriously threaten people’s lives and health.Virus detection is an essential prerequisite for prevention and treatment of viral infections.Common virus detection methods mainly include polymerase chain reaction(PCR)and enzyme-linked immunosorbent assay(ELISA).These methods show excellent performance in virus detection and provide accurate and reliable results.However,they still have some limitations.For example,PCR relies on sophisticated temperature control equipment and professional operators.The operation steps are complex and the detection time takes several hours.ELISA requires expensive antibodies,which greatly increases the cost of detection.The detection process needs cumbersome separation and washing steps,which are prone to cross contamination.These limitations are not conducive to their work in low resource allocation areas and difficult to realize point-of-care testing.Therefore,it is urgent to develop high sensitivity,simple operation,and rapid response methods for the diagnosis of viral infectious diseases to break through the limitations of existing detection technologies on personnel and places.CRISPR/Cas systems,especially Cas12 a and Cas13 a,possess the specific activation of nucleic acid targeting and unique trans-cleavage ability,which has become a promising detection technology in the field of molecular diagnosis.Based on this,CRISPR/Cas12 a,CRISPR/Cas13 a and isothermal amplification technology are combined to establish a simple,rapid and highly sensitive method for viral nucleic acid detection.In addition,a generic micro RNA detection method has been developed.The specific research contents are as follows:(1)A one-step detection method for SARS-Co V-2 N plasmid was established based on RPA and CRISPR/Cas12 a.All components of RPA and CRISPR/Cas12 a reaction were assembled in an EP tube and reacted at constant temperature.Based on the fact that CRISPR/Cas12 a system does not require PAM sequence when recognizing single-stranded DNA,two cr RNAs were introduced.They could specifically bind to the single-stranded sequence appearing in the process of RPA amplification,which activated the trans-cleavage activity of Cas12 a,and cut the C-rich signal reporter probe to generate signals.At the sa me time,the amplification product of RPA started a new round of cycle,and so on.Cas12 a could be activated continuously in such repeated reaction,thus signal amplified.Compared with a single cr RNA,the use of two cr RNAs significantly improved the sensitivity of detection.This method was simple and could be completed in one step.And the detection time is short,which only took 30 minutes to complete the detection of the target with good selectivity.In addition,the practicability of the method was verified by the spiking experiment.(2)The detection method of SARS-Co V-2 RNA was proposed based on CRISPR/Cas13 a trans-cleavage triggered CHA.A hairpin reporter probe was designed as the CRISPR/Cas13 a trans-cleavage substrate.When Lwa Cas13 a protein activated,the hairpin reporter probe was induced to cleave and release the primer.After adding M1 and M2 into the reaction system,the primer triggered the CHA reaction,amplifying the detection signal.The detection limit of CRISPR/Cas13 a against the target RNA was 10 p M.When CRISPR/Cas13 a combined with CHA,the detection of the target RNA was as low as 84 a M,indicating that CRISPR/Cas13 a combined with CHA was able to reduce the detection limit by about 5 orders of magnitude.Due to the programmability of cr RNA,this method was successfully applied to the detection of Echovirus RNA,providing a general detection method.(3)We established a one-step detection method for Echovirus RNA based on CRISPR/Cas13 a and CHA.Based on the work in the previous chapter,Lwa Cas13 a was replaced by Lbu Cas13 a with higher trans-cleavage activity.And the reaction buffer was optimized to make the components of CRISPR/Cas13 a and CHA reactions in the same container,which simplified the nucleic acid analysis operation.The one-step method has excellent analytical performance,and the detection sensitivity was about 4 orders of magnitude higher than that of CRISPR/Cas13 a system.The results of clinical samples were consistent with those of the standard method RT-q PCR.Because of its simple operation,short detection time and high sensitivity,the one-step method has potential application value in clinical diagnosis.(4)Establishing an amplification-free detection method for monkeypox virus DNA based on Cas12 a and multiple cr RNAs.Based on the B6 R gene of monkeypox virus,four cr RNAs were designed.And multiple CRISPR/Cas12 a could simultaneously bind to different sites on the same target sequence,which activated more Cas12 a and improved the efficiency of trans-cleavage.Compared with a single cr RNA,the combination of multiple cr RNAs not only shortened the analysis time,but also improved the sensitivity by 4-27 times.This amplification-free detection method provided a new idea for the diagnosis of monkeypox.(5)A micro RNA detection method was developed based on EXPAR and CRISPR/Cas12 a.Micro RNA-27 a acted as a primer to initiate EXPAR,which generated a large number of activators through polymerization reactions and displacement reactions.These activators triggered the trans-cleavage of CRISPR/Cas12 a,cleaving signal reporter probe to obtain the detection signal.The detection sensitivity of this method was as low as 90 a M.It was able to distinguish single base mismatches.And the detection results of micro RNA-27 a in different cell lines by this method were consistent with those of RT-q PCR.More importantly,through the flexible design of EXPAR template,the proposed method was applicable to the detection of arbitrary micro RNAs,providing a general strategy for molecular diagnosis.In summary,this paper constructed a series of nucleic acid detection methods by coupling CRISPR/Cas with isothermal amplification technology.The results showed that these methods have the advantages of simple,rapid,ultra-sensitive and high specificity,and could be used for the detection of actual samples.Therefore,these methods had great potential in the field of molecular diagnosis. |
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