Establishment Of RNA Virus Detection Method Based On Crispr/Cas13 And Nanopore Sequencing

Background:In the past two decades,public health emergencies have occurred frequently.The epidemics caused by RNA viruses such as SARS coronavirus,Middle East respiratory syndrome coronavirus,and 2019 novel coronavirus have posed a great threat to human life and health.The high variability of RNA virus genomes has brought great challenges to virus detection.Rapid and accurate identification of pathogens is the key to overcoming the epidemic.Nucleic acid sequencing technology,especially the nanopore sequencing technology-developed in recent years,plays an increasingly important role in the discovery of pathogens in major epidemics.However,problems such as low virus titer,complex sample background and interference of host nucleic acids often lead to miss detection,which is the technical bottleneck of virus detection technology.The CRISPR/Cas system is characterized by sequence-dependent recognition,which detects specific target sequence and enables multiple pathogen detection.Cas13d can specifically bind target RNA through single-stranded guide RNA(Cr RNA).This study aims to use the characteristics of Cas13d coupled with nanopore sequencing to achieve targeted enrichment of RNA viruses and establish a rapid and accurate RNA virus detection method.Purpose:Aiming at the needs of RNA virus detection under complex background conditions,this study combines CRISPR/Cas13d and nanopore sequencing technology to establish a virus detection method that can target and enrich the RNA virus genome.Methods andResults:We established a virus nucleic acid detection method based on CRISPR/Cas13d targeting technology and Nanopore sequencing technology,and preliminarily verified it using virus mimics containing 2019 novel coronavirus sequences.In this method,mutated Cas13d and Cr RNA specifically bind to viral RNA in mixedRNA samples,and the d Cas13d-target sequence complex is enriched by biotin-streptavidin system.The enriched viral RNA is subjected to nanopore sequencing technology to obtain nucleic acid sequence,and further identified by bioinformatics comparison.In order to establish this method,we first designed four Cas13d protein mutants with shear active domain(HEPN)mutations.Through shear activity analysis and affinity constant determination,we found that the shear activity of the mutant H863A was completely destroyed,and its affinity with the designed target sequence was equivalent to that of the wild type.We designed and synthesized specific Cr RNAs for 2019 novel coronavirus,and constructed virus mimics containing corresponding virus sequences for method evaluation.The preliminary method verification results show that the target RNA was effectively enriched can.In addition,another part of my work is to establish a field detection method for 12gastrointestinal pathogens based on multiplex PCR and nanopore sequencing technology.We designed specific primers for 12 kinds of digestive tract viruses such as adenovirus,norovirus,astrovirus,Parechovirus,Enterovirus,rotavirus and Sapovirus,verified the specificity of primers,optimized the conditions of multiplex PCR amplification,and constructed a rapid library construction and sequencing system.Finally,the detection sensitivity of pathogens was evaluated.The results showed that the established multiplex PCR detection system for 12 digestive tract pathogens could effectively amplify all target sequences.The sensitivity of this method was analyzed by using mixed DNA mimics of12 pathogens and mixedRNA samples of three rotaviruses A,B and C.The results showed that the detection sensitivity of this method could reach 10~3 copies/m L,which was comparable to that of real-time quantitative PCR.Conclusion:In this thesis,two virus detection methods for targeted enrichment of viral genomes were established.By reducing the influence of background nuclear acid,the sensitivity and specificity of virus detection were improved.At the same time,the sequence information of pathogens could be obtained,which will be very important for reducing false positivity pathogen tracing.In summary,the method established in this paper can provide powerful support for pathogen detection in public health emergencies.