Research On Pathogen Multiple Detection Technology Based On Nanopore Sequencing

BackgroundDisease outbreaks caused by pathogen infection threaten global public health security seriously,such as novel coronavirus pneumonia(Coronavirus disease 2019,COVID-19),Middle East respiratory syndrome(MERS),Ebola virus disease and Crimean Congo hemorrhagic fever(CCHF),etc.Commonly used pathogen detection methods include immunological methods based on the principle of antigen-antibody detection and molecular biological methods based on nucleic acid amplification.Although the above methods have greatly helped the treatment of related diseases,it is difficult to obtain specific sequence information,and the specificity requirements of primer design are really high,which may easily cause the risk of false positive detection of pathogens.In order to solve the above technical difficulties,we adopted two technical strategies of CRISPR-Cas9 and multiplex PCR.By enriching target sequence fragments and using nanopore sequencing technology to sequence and analyze the target sequence,we established an accurate,specific and applicable on-site and home-operated technical methods to achieve accurate and specific identification and detection of pathogens,curb the spread of the epidemic as soon as possible,and reduce the harm to human life,health and safety.The CRISPR-Cas9 system is a widely used gene editing tool with the ability to specifically recognize and cut target sequences.The CRISPR-Cas9 system consists of sg RNA and Cas9.CRISPR RNA and tracr RNA are combined to form sg RNA.Under the guidance of sg RNA,Cas9 protein recognizes the PAM site of the target sequence and cuts the target site precisely,so as to realize the target sequence purification and enrichment of fragments.Polymerase chain reaction(PCR)is a molecular biology technique used to amplify specific DNA fragments in vitro.Multiplex PCR technology is based on PCR technology,introducing multiple pairs of specific primers into the same reaction system to amplify the target sequence,which can simultaneously amplify a variety of different pathogens.Nanopore sequencing technology is an emerging sequencing analysis technology in recent years.Its principle is that when the DNA sequence passes through the nanopore protein embedded at both ends of the biological membrane,the change of the protein structure will cause the change of the ion concentration at both ends of the biological membrane,and the potential difference will lead to the change of the real-time current signal.The data terminal can decode it into a base sequence signal in real time,and realize the instant reading of the pathogen gene sequence.At the same time,the nanopore sequencing Min ION platform used for detection is a portable handheld system suitable for on-site detection.Therefore,two strategies of target sequence enrichment,CRISPR-Cas9 and multiplex PCR,were adopted and combined with nanopore sequencing technology respectively to establish accurate,specific and rapid methods for multiplex pathogens detection.Research contents1.Establishment of pathogen multiple detection method based on CRISPR-Cas9combined with nanopore sequencing technologyFirstly,Staphylococcus aureus enterotoxin base(seb),Clostridium botulinum toxin A gene(Bo NT＿A)and Vibrio parahaemolyticus hemolysin gene(tdh)were selected as target genes,corresponding cr RNA were design.RNP complexes were produce by assembling cr RNA with Cas9 protein.Secondly,cr RNA,Cas9 protein,shearing time,etc.were optimized respectively,and then the sensitivity of the system was evaluated.Lastly,the CRISPR-Cas9-nanopore system were tested for detection of 29 respiratory pathogens.For this purpose,2 cr RNAs for each selected pathogen were design and synthesized,and multiple detection were evaluated using DNA templates.3.Establishment and evaluation of a detection method for respiratory pathogens based on multiplex PCR combined with nanopore sequencing.Firstly,specific primers for 29 respiratory pathogens were designed to amplify pathogen target genes by multiplex PCR.The specificity and compatibility of the primers were evaluated.Secondly,the multiplex PCR system was optimized by adjusting the primer concentration and annealing temperature.Lastly,the sensitivity was evaluated with the synthetic DNA template.Results1.For a single virulence gene,the target sequence can be successfully detected after Cas9-mediated specific cleavage.After evaluating the target-cutting efficiency of each cr RNA,two with the highest efficiency were selected for multiplex detection.Target sequences of all three virulence genes can be successfully detected with substantial amount of human genomic DNA background.The cutting efficiency of Hi Fi Cas9 is better than that of Cas9.DNA fragmentation of the sample can effectively improve the detection efficiency.Sequencing of the background nucleic acid can be successfully reduced using the CRISPR-Cas9-nanopore sequencing system.With or without Cas9-mediated targeted cutting,the sequenced human genomic DNA reads decreased from 98.68%to 13.88%,and the reads of target sequence increased from 2.5‰to 58.02%.The target gene detection sensitivity of the CRISPR-Cas9-nanopore sequencing system is 1×10~9copies/m L.Multiple detections were carried out for 29 respiratory pathogens,and preliminary results showed that the gene sequences of 15 pathogens could be detected in a single experiment.2.The multiplex PCR-nanopore sequencing system were evaluated for detection of29 respiratory pathogens.The optimal primer concentration is 0.1μM with detection sensitivity of 1×10~4 copies/m L,25 kinds of respiratory pathogens can be stably detected.ConclusionBased on nanopore sequencing technology,this study established a technical system for the detection of multiple pathogens.Two strategies of pathogen sequence enrichment were adopted,CRISPR-Cas9 and multiplex PCR.With nanopore sequencing,accurate pathogen nucleic acid sequence information can be obtained and false positive can be effectively avoided.Through combining efficient enrichment and sequencing technologies,two rapid,accurate and specific pathogen detection methods have been established,which,provide new strategies for rapid on-site detection of pathogens.