Establishment Of CRISPR-based Virus Detection Method For Severe Fever With Thrombocytopenia Syndrome

Objective: Fever with thrombocytopenia syndrome is a new infectious disease first reported in China in 2007.At present,SFTSV infection has been reported in more than26 provinces.As an emerging tick-borne infectious disease,the incubation period is not clear,and it has a wide spread risk.The clinical manifestations mainly include fever,gastrointestinal symptoms,and critical illness.It can die from shock and multiple organ failure.The mortality rate is as high as 30%.It is a natural focal disease with a high mortality rate found in China in recent years.It has become a serious public health security problem in China and even in the world.Due to the lack of specificity and complex symptoms,there is currently no effective prevention,control and treatment.Therefore,early diagnosis and timely blocking of transmission are the key.In recent years,the lack of the latest detection technology.Fast,efficient and accurate virus detection methods are urgently needed.CRISPR is widely used in gene editing,disease model construction,gene targeted therapy and other fields.Due to its high specificity and sensitive detection rate,CRISPR technology has reached the detection of low to single copy nucleic acid in pathogen detection,providing a new technical solution for rapid detection of pathogens.The subsequent development of nucleic acid detection technology-SHERLOCK : combined with CRISPR-Cas13 a and recombinase-mediated isothermal nucleic acid amplification technology,the results were visualized by colloidal gold immunochromatographic test strips.It can specifically detect viral nucleic acids and can detect a series of clinically relevant low-frequency tumor-related mutations.In this study,based on Sherlock detection technology,a nucleic acid detection method was established for the S gene fragment of SFTSV,and the sensitivity and specificity of the detection method were evaluated in order to improve the detection ability of SFTSV.Methods: 1.The whole genome sequence of SFTSV was downloaded from the official website of the National Biotechnology Information Center for conservative analysis,virus positive plasmid was constructed,RAA primers and cr RNA were designed using Primer and Oligo.2.Induced expression and purification of Cas protein,screening cr RNA.3.The sensitivity and specificity of the detection method were evaluated by CRISPR fluorescence and test paper,and the laboratory RT-PCR detection method was compared.Results: 1.The virus positive plasmid was successfully constructed by selecting the conserved gene fragment,and a pair of RAA constant temperature amplification primers and 5 groups of cr RNA were designed.2.Cas protein expression and purification of 0.25 mg,screened a group of cr RNA.3.The CRISPR detection method for SFTSV was successfully constructed,with a sensitivity of 1copy/μL,good specificity evaluation,and a reaction time of up to 90 min,which was better than 150 min of RT-PCR.Conclusion: In this study,based on CRISPR technology,CRISPR-cas13 a,RAA and colloidal gold immunochromatographic test strip were combined for the first time to establish a simple,rapid,highly sensitive and highly specific nucleic acid detection method for SFTSV,which provided new ideas for rapid on-site detection in the future.