Research On New Technology For Detection Of Food-borne Pathogenic Microorganisms Based On CRISPR

Objective:Food-borne diseases caused by pathogenic microorganisms are the primary public health problem in the world,which seriously threatens the safety of human life and property.Common pathogenic microorganisms include bacteria,viruses and fungi.The purpose of this study is to establish a new detection method based on CRISPR technology for hepatitis E virus type IV and Vibrio parahaemolyticus,which are prevalent in China,and provide technical means for the prevention and control of related diseases.Methods:1.Establishment and application of the RPA-CRISPR detection method for hepatitis E virus:The sequence comparison software MEGA 7.0 was used to compare the whole gene FASTA sequences of hepatitis E virus type IV from different sources in the NCBI database to screen out its common conservation sequence and construct plasmid standards.According to the characteristic that Cas12a can only recognize T-rich PAM sequences,we selected the sequence of 20bp in length and qualified as the detection target from the conserved sequences screened.The cr RNA was designed and prepared according to the target sequence,and the single-stranded DNA probe was designed and synthesized.Then,the Cas12a core cutting system for hepatitis E virus was initially established,and its feasibility and sensitivity were tested.According to the primer design instruction manual of Twist Amp Company,the RPA primer pair HEV-F1/HEV-R1 was designed in the range of the conserved sequence of hepatitis E virus,and the amplified product was purified and electrophoresed to verify its amplification performance.Subsequently,an RPA-CRISPR detection method for hepatitis E virus was established by adding the RPA amplification product to the Cas12a core cleavage system,and by optimizing the components in the system,the detection results were displayed in three different visualization schemes,respectively:fluorescence quantitative PCR instrument method,naked eye observation method under blue light and flow chromatographic test strip method.The sensitivity of the RPA-CRISPR method was then tested with the above three visualization schemes,and the specificity of the RPA-CRISPR method was tested with the fluorescence quantitative PCR instrument method.After establishing the gold standard quantitative PCR detection method for hepatitis E virus,the RPA-CRISPR fluorescence quantitative PCR instrument method and quantitative PCR method were used to detect 30 artificially diluted plasmid templates with different concentrations.Correlation analysis was carried out on the detection results of the two methods to test the consistency of the results of the two methods.Finally,30 stool samples from patients with suspected hepatitis E infection retained in our laboratory were tested using three visualization schemes of RPA-CRISPR method and quantitative PCR method to test the practical ability of RPA-CRISPR method.2.Study on efficient enrichment method of hepatitis E virus based on PRTM-G-Fe₃O₄:G-Fe₃O₄ composite was prepared by solvothermal method and modified with highly positively charged protamine to prepare PRTM-G-Fe₃O₄.The composites were characterized by TEM analysis,XPS analysis,XRD analysis,FTIR analysis,zeta potential analysis,N₂ adsorption and desorption analysis and magnetic property analysis.Subsequently,an enrichment system for hepatitis E virus was established,and the conditions were optimized from the three aspects of material concentration,adsorption time and p H value of the system to improve the enrichment efficiency.Finally,the system was used to enrich for hepatitis E virus in fecal samples and detect it using the RPA-CRISPR method established in Chapter 1 to evaluate its utility.3.Establishment and application of RPA-CRISPR detection method for Vibrio parahaemolyticus:The FASTA sequences of the tdh and trh genes of Vibrio parahaemolyticus from different sources in the NCBI database were compared using the sequence alignment software MEGA 7.0 to screen out their consensus conserved sequences and construct plasmid standards.According to the characteristic that Cas12a can only recognize T-rich PAM sequences,we selected 20bp in length,qualified sequences from the conserved sequences of tdh and trh genes as detection targets.The cr RNA was designed and prepared according to the target sequence,the single-stranded DNA probe was designed and synthesized,and then the Cas12a core cleavage system for the tdh and trh genes of Vibrio parahaemolyticus was established respectively,and its sensitivity was tested.According to the primer design instruction manual of Twist Amp company,the RPA primer pairs F1/R1 and F2/R2 were designed in the range of the conserved sequence of tdh gene,RPA primer pairs F3/R3,F4/R4 and F5/R5 were designed in the range of conserved sequences of trh gene,and the amplified products were purified and electrophoresed to verify their amplification performance.Then,the RPA-CRISPR detection method for Vibrio parahaemolyticus tdh and trh genes was established according to the method shown in Chapter 1,and the detection results were displayed by fluorescence quantitative PCR instrument method and naked eye observation method under blue light,respectively.The sensitivity of the RPA-CRISPR method was then tested with the above two visualization schemes,and the specificity of the RPA-CRISPR method was tested with the fluorescence quantitative PCR instrument method.After establishing the gold standard quantitative PCR detection methods for Vibrio parahaemolyticus tdh and trh genes respectively,10 samples of suspected Vibrio parahaemolyticus retained in our laboratory were detected by two visualization schemes of RPA-CRISPR method and quantitative PCR method,respectively.According to the test results,the No.6 sample with both positive gene test results was selected,its bacterial solution was serially diluted,and the two visualization schemes of the RPA-CRISPR method were used for detection to test the sensitivity of the method to the actual bacterial solution.Finally,according to the above test results,a bacterial suspension of positive samples with a concentration of 10² CFU/g was prepared to prepare spiked fresh shrimp samples,which were detected by the RPA-CRISPR blue light observation method to test the practical ability of the RPA-CRISPR method.Results:1.We have established a quantitative PCR detection method for hepatitis E virus,which can detect the plasmid template with a concentration as low as 10^-17 M.The linear regression coefficient of the standard curve is 0.997,and the amplification efficiency is 96.752%.Through component integration,we initially established a Cas12a core cleavage system for hepatitis E virus and evaluated its feasibility.We found that only when the four main components of the system Cas12a,cr RNA,target DNA and ss DNA reporter are complete,The system will complete the detection of the target and display the fluorescent signal as expected,and then perform a sensitivity test on it.The results show that the detection limit of the Cas12a core cleavage system is 10n M.In view of the conserved sequence of hepatitis E virus,we designed the RPA primer pair HEV-F1/HEV-R1,and carried out RPA amplification with 1μM plasmid standard as the template.After purification,the product was subjected to 1.5%agarose gel electrophoresis.The results showed that the primer pair HEV-F1/HEV-R1successfully amplified a band with a length of 186bp,which was in line with the expectation.By adding the RPA amplification product to the Cas12a core cleavage system,we established an RPA-CRISPR detection method for hepatitis E virus,and by optimizing the components in the system,the detection results can be displayed in three different visualization schemes.We tested it for sensitivity and specificity,and the results showed that the detection limit of this method is 10^-17 M,and the specificity is good,it will not cross-react with other kinds of viruses.In order to explore the correlation between the detection results of the quantitative PCR method and the RPA-CRISPR method,we used two methods to detect 30 artificial plasmid samples with different dilution degrees.The results showed that the detection results of the two methods were consistent,and the two results shows a good positive correlation.Finally,in order to explore the practical ability of RPA-CRISPR method,we used quantitative PCR method and RPA-CRISPR method to detect 30 stool samples of patients suspected of hepatitis E infection retained in our laboratory,respectively.The results showed that the two methods yielded the same test results:out of 30 samples,2 were positive and the rest were negative,which also showed that the method had good practicability.2.Characterization of various projects on the composite material G-Fe₃O₄ before protamine modification and the composite material PRTM-G-Fe₃O₄ after modification:The results of TEM analysis showed that Fe₃O₄ nanoparticles were uniformly dispersed on the surface of the wrinkled graphene,and after modification,the oval particles suspected of protamine were adsorbed on the surface of the composite material;the results of energy spectrum analysis and XPS analysis showed that G-Fe₃O₄ is mainly composed of four elements,Fe,O,C and N,while PRTM-G-Fe₃O₄ is mainly composed of five elements of Fe,O,C,N and S.The extra N and S elements in the composite material come from protamine;XRD analysis and FTIR analysis proved the successful preparation of G-Fe₃O₄ and the successful modification of PRTM-G-Fe₃O₄ from the crystal structure and surface chemical composition,respectively;the results of zeta potential analysis showed that at different p H values,the zeta potential of PRTM-G-Fe₃O₄ is higher than that of G-Fe₃O₄,the isoelectric point of G-Fe₃O₄ is 8～9,and the isoelectric point of PRTM-G-Fe₃O₄ is greater than 10,which means that the composite material carries more positive charges after modification,which is also more conducive to the adsorption of viruses;the results of N₂ adsorption and desorption analysis showed that PRTM-G-Fe₃O₄ had a porous structure with a specific surface area of 39.983 m²/g;the results of magnetic property analysis showed that PRTM-G-Fe₃O₄ has good superparamagnetic properties,and its magnetic saturation is43.4 emu/g.The results of the optimization of the enrichment system conditions showed that for 10⁶ copies of hepatitis E virus,the concentration of PRTM-G-Fe₃O₄ composite material in the enrichment system was 0.2 mg/m L,the adsorption time was 30 min,and the p H value of the system was defaulted to 7,the optimum enrichment efficiency was95.7%.Finally,we combined the enrichment system with the RPA-CRISPR detection method established in Chapter 1 to detect virus suspensions with different concentrations,the results showed that when faced with samples with complex components and relatively low virus concentrations,an efficient enrichment process can improve the detection capability of subsequent detection methods.3.We first established a quantitative PCR detection method for the two genes of Vibrio parahaemolyticus.The results show that:for the tdh gene,this method can detect the plasmid template with a concentration as low as 10^-17 M,and the linear regression coefficient of the standard curve is 0.988,the amplification efficiency was 83.736%;for the trh gene,this method could also detect the plasmid template with a concentration as low as 10^-17 M,the linear regression coefficient of the standard curve was 0.983,and the amplification efficiency was 83.036%.We established the Cas12a core cutting system for the tdh and trh genes of Vibrio parahaemolyticus and tested its sensitivity.The results showed that the detection limit of the Cas12a core cutting system was 10 n M for both genes.Within the range of the conserved sequences of the two genes,we designed RPA primer pairs F1/R1 and F2/R2 for the tdh gene,and the amplicon lengths were213bp and 247bp,respectively;we designed RPA primer pairs F3/R3,F4/R4 and F5/R5for the trh gene,and the amplicon lengths were 215bp,228bp and 233bp,respectively.Using 1μM plasmid standard as a template,RPA amplification was performed,and the product was purified and subjected to 1.5%agarose gel electrophoresis.The results showed that the five pairs of primers successfully amplified the expected bands.Then we established the RPA-CRISPR detection method for the tdh and trh genes of Vibrio parahaemolyticus according to the method shown in Chapter 1,and realized the detection results in two different visualization schemes,and tested their sensitivity.The results showed that:for the tdh gene,the primer pair F1/R1 has the best performance,and it only takes 25 minutes to detect the plasmid template with a concentration as low as 10^-18 M;for the trh gene,the primer pair F5/R5 has the best performance,and it only takes 30 minutes to detect the plasmid template with a concentration as low as 10^-18 M.The specificity test showed that the method has good specificity and will not cross-react with other types of bacteria.We used quantitative PCR method and RPA-CRISPR method to detect 10 suspected Vibrio parahaemolyticus positive samples retained in our laboratory respectively,and the two methods obtained consistent detection results:out of 10 samples,7 samples were positive,3 negative samples,including 5 tdh gene positive samples and 6 trh gene positive samples.Subsequently,we selected sample No.6,which was positive for both genes,and carried out gradient dilution for it.The RPA-CRISPR method was used for detection.The results showed that the method could detect bacterial culture with a concentration as low as 10² CFU/g.Finally,we selected 7positive samples detected in the previous stage,prepared bacterial solutions with a concentration of 10² CFU/g respectively,prepared spiked fresh shrimp samples,and detected them using the RPA-CRISPR method,and obtained the same results as the previous ones.The method can be used to detect Vibrio parahaemolyticus in food samples,and has good practicability.Conclusion:In this study,an RPA-CRISPR detection method for hepatitis E virus was established.The sensitivity of the method can reach 10^-17 M,the specificity is good,the results are visible,and large-scale experimental equipment is not required.Compared with traditional detection methods,it is more suitable for on-site detection.In the detection of plasmid templates of different concentrations,the detection results of this method have a good positive correlation with the quantitative PCR method;in the detection of fecal samples,the detection results of this method are completely consistent with the quantitative PCR method,indicating that its usability is good.Subsequently,we established an efficient enrichment system for hepatitis E virus based on PRTM-G-Fe₃O₄.When the concentration of composite material in the system is 0.2mg/m L,the adsorption time is 30 min,and the p H value of the system is 7 by default,the best enrichment efficiency is 95.7%.And the relevant experimental results also showed that the efficient enrichment of the virus in the early stage can improve the detection ability of the subsequent detection method.Finally,we established the RPA-CRISPR detection method for Vibrio parahaemolyticus tdh,trh genes according to the method in Chapter 1.This method can detect plasmid templates with concentrations as low as 10^-18 M within 30 min,and also has good specificity.In the detection of actual bacterial samples,the results of this method are consistent with the quantitative PCR method,and this method can detect bacterial culture with a concentration as low as 10² CFU/g.Finally,we used this method to detect artificially spiked shrimp samples with a concentration of 10² CFU/g,and we could also get accurate results,indicating that the method is practical.