Construction Of Whole-genome Database Of Pathogenic Listeria And Research On Novel Techniques Of CRISPR/Cas-based Nucleic Acid Detection

Listeria monocytogenes and Listeria ivanvii are two common foodborne pathogens of Listeria spp.,especially L.monocytogenes,which can cause many serious diseases such as meningitis and sepsis after infection,with a mortality rate of up to 30%of susceptible people.Nucleic acid detection methods based on target sequence amplification have been widely used in the identification and typing of Listeria due to their advantages of speed and sensitivity.However,the number of targets for Listeria detection is limited,and may be mutated or deleted by environmental stress in the process of genetic evolution.Therefore,it is of great significance to excavate new specific molecular detection targets related to Listeria,and on this basis to establish sensitive and specific nucleic acid detection methods.In this study,the whole-genome database and target mining platform of Listeria were constructed using second-generation sequencing and bioinformatics technology,and new species-specific and serotypes-specific targets related to common Listeria spp.and L.monocytogenes serotypes were obtained.Based on these new molecular targets,multiplex PCR(m PCR),PCR/RAA-CRISPR/Cas12a fluorescence detection(PCR/RAA-Cas12a FDet),RAA-CRISPR/Cas12aelectrochemicaldetection(RAA-E-CRISPR),and RAA-CRISPR/Cas13a-based centrifugal microfluidic chip were established,respectively.The main contents and results of this study are as follows:1.The“foodborne L.monocytogenes genome database”was constructed based on our previous investigations of L.monocytogenes contamination in 4300 food samples and 925isolates across China from 2011 to 2016,and 841 whole-genome sequences of L.monocytogenes sequenced by our team.Combined with the whole-genome sequences of Listeria in NCBI and pan-genome analysis technology,the molecular detection target genes mining platform for Listeria common species-specific and L.monocytogenes serotypes-specific genes was established.The results showed that the L.monocytogenes isolates belonged to five serotypes:1/2a,1/2b,1/2c,4b,and 4c.Listeria monocytogenes had an open pan-genome and contained 528 core genes,which could be divided into three lineages:I,II and III.By using the target mining platform,50 and 37 candidate targets related to Listeria common species and L.monocytogenes serotypes were obtained.2.The specificity and sensitivity of the candidate targets were further screened by PCR,a total of 16 targets for Listeria common species were obtained,including one target for each of Listeria spp.,L.monocytogenes,and L.welshimeri;three targets for each of L.innocua and L.ivanvii;two and five targets of L.grayi and L.seeligeri,respectively.A total of 14 targets related to L.monocytogenes serotypes were also obtained,including one,two,three for lineage I(1/2a-3a-1/2c-3c),II(1/2b-3b-4b-4d-4e-4ab-7),and III(4a-4c);two,one,one,and one for serogroup I.1(1/2a-3a),I.2(1/2c-3c),II.1(4b-4d-4e-4ab),and II.2(1/2b-3b-7);one and two for serotype 4a and 4c,respectively.The PCR sensitivity of some targets was up to10~2CFU/m L.Biological function prediction analysis of specific targets revealed that the proteins encoded by these genes were related to host infection,carbohydrate transport,and adaptability to different growth environments,however,most of the gene-encoded proteins were hypothetical proteins.3.Based on the new target of Listeria species,the m PCR assay was established for the detection of L.monocytogenes,and L.ivanvii.The detection limits in pure culture medium were 2.0×10~3CFU/m L and 3.4×10~3CFU/m L,respectively.At the same time,based on these serotype-related specific new targets,two“lineage”m PCR assays were developed for rapid identification of L.monocytogenes serogroups I.1,I.2,II.1,II.2 and III.The detection limits of lineage I and lineage II&III m PCRs were 0.771 pg/?L and 1.76 pg/?L genomic DNA,respectively.All the above m PCR methods showed good specificity to target bacteria.The established m PCR was employed for analyzing 129 Flammulina velutipes(F.velutipes)plants samples,the detection rates of Listeria spp.and L.monocytogenes was 58.1%and41.1%,respectively.In addition,24 samples were selected for serotype detection,four serogroups I.1,I.2,II.1,II.2 were identified.The results of the above m PCR assays were highly consistent with those of the traditional culture methods.4.The PCR/RAA-Cas12a FDet method was established for detection of L.monocytogenes serotype 4c by using the“collateral”cleavage activity of CRISPR/Cas12a.In this system,the PCR or RAA reaction reagents and Cas12a/cr RNA cleavage reagents were encapsulated in the reaction tube.After amplification,Cas12a FDet analysis was performed by centrifugation,which effectively avoided aerosol pollution.The results showed that both PCR/RAA-Cas12a FDet showed high specificity for detection of serotype 4c strains,and the detection limits of PCR-Cas12a FDet and RAA-Cas12a FDet were 34 CFU/m L and 1.4×10~2CFU/m L in pure culture,respectively.In genomic DNA and spiked samples,the detection limits of RAA-Cas12a FDet method were 0.64 amol/L and 1.4×10~3CFU/g fresh grass carp.5.The“collateral”cleavage activity of CRISPR/Cas12a was innovatively introduced into the electrochemical platform,and the RAA-E-CRISPR method was established for the detection of L.monocytogenes.In this method,ss DNA reporter modified with methylene blue(MB)(MB-ss DNA)was assembled on the surface of the gold electrode.In the presence of the target bacteria,the“collateral”cleavage ability of Cas12a is activated by RAA amplification products,cleaving the MB-ss DNA reporter off the electrode surface.The target bacteria can be detected by measuring the current signal before and after the introduction of the target nucleic acid to the sensor.The results showed that this method showed excellent specificity,with the detection limits of 0.68 amol/L of genomic DNA,26 CFU/m L of L.monocytogenes in pure cultures,9.4×10~2CFU/g of L.monocytogenes in spiked F.velutipes,respectively.Compared with the RAA-Cas12a FDet,the RAA-E-CRISPR can potentially improve the sensitivity by 10-fold.6.To achieve one-step,multiplex detection,a centrifugal microfluidic chip based on RAA-CRISPR/Cas13a was established for detection of L.monocytogenes and L.innocua.During microfluidic chip fabrication,the RAA-CRISPR/Cas13a reagents were preloaded into the reaction chambers.In this way,the operator only needs to add the nucleic acid sample into the sample chambers,and the amplification,detection and data readout can be done automatically on the microfluidic device.The results showed that this method showed excellent specificity,the detection limits of L.monocytogenes and L.innocua were 0.40amol/L and 0.74 amol/L of genomic DNA,40 CFU/m L and 26 CFU/m L for pure culture without enrichment,3.4×10~3CFU/g and 2.6×10~2CFU/g in spiked F.velutipes,respectively.In summary,the whole-genome database and target mining platform of Listeria were constructed in our study,and a batch of specific molecular detection new targets related to Listeria common species and L.monocytogenes serotypes with China's independent intellectual property rights were obtained,which effectively made up for the shortcomings of existing targets.In addition,the“collateral cleavage”activity of CRISPR/Cas was innovatively introduced into electrochemical DNA biosensor platform and centrifugal microfluidic chip,and a series of rapid,sensitive and specific nucleic acid detection methods based on CRISPR/Cas were established with new targets,which improves the detection performance of the existing CRISPR/Cas nucleic acid detection system and broadens its application,providing basic data and technical support for risk identification,traceability and rapid detection of L.monocytogenes.