High-Throughput Detection Of Four Common Foodborne Pathogenic Bacteria Based On The Mag-Cds-MPCR-CE

Objective:In recent years,there have been frequent occurrences of adverse food safety incidents worldwide,which have seriously affected the economic conditions and social stability of many countries,including S.typhimurium,S.aureus,L.monocytogenes,and E.coli O157:H7 pollution.Bacterial food poisoning accidents caused by food are more common.Food-borne pathogen infections often cause clinical symptoms such as vomiting,fever,abdominal pain,diarrhea,and shock,in severe cases,it can even lead to circulatory failure.At present,the laboratory detection methods for the above-mentioned food-borne pathogens mainly include traditional separation and culture biochemical identification methods,immunological detection methods and molecular biological detection methods.However,these methods are not only complicated and time-consuming,but also have low detection sensitivity and specificity,and require high experimental conditions and technical personnel.Therefore,the establishment of a simple,fast,sensitive and accurate high-throughput detection method is necessary to prevent and control the food safety problems caused by the above four food-borne pathogens.This subject established a new high-throughput rapid detection method for food-borne pathogens by combining magnetic separation technology,multiple PCR technology and capillary electrophoresis detection technology,and evaluated the established detection method,which is safe for food.Supervision,prevention and control provides certain technical support.Methods:1.Preparation and characterization of Mag-CdsThe solvothermal method was used to synthesize Fe₃O₄nanoparticles with surface modified carboxyl groups,and chitosan was used as a carbon source to coat Fe₃O₄nanoparticles to prepare Mag-Cds capable of simultaneously adsorbing and separating Gram-negative and Gram-positive bacteria.Use biological transmission electron microscope,Fourier infrared spectrometer and other equipment to observe the prepared functionalized carboxyl magnetic beads and immunomagnetic beads to characterize the size,morphology and performance.2.Determination of Mag-Cds magnetic enrichment efficiency for four food-borne pathogensAfter Mag-Cds is successfully prepared,S.aureus is used as the target bacteria,the dosage of Mag-Cds is optimized,and the optimal dosage of Mag-Cds is determined.S.typhimurium,S.aureus,L.monocytogenes and E.coli O157:H7 were used to determine the enrichment efficiency.3.Establishment and optimization of MPCR systemSearch databases and related literature,refer to published specific genes or targets and NCBI database for BLAST alignment and preliminary screening of corresponding conserved sequences and primers of foodborne pathogens,and use software such as Oligo to exclude the formation of dimers and hairpins After confirming through experiments that all four pathogens can be successfully amplified,establish a multiplex PCR system.Set different gradients for key factors such as primer concentration,d NTPs,Mg²⁺,HS DNA polymerase,and annealing temperature,and pre-denature in the PCR machine.95℃,5 min,94℃denaturation1 min,55℃annealing for 30 s,72℃renaturation 1 min,after 30 cycles,extension72℃,10 min.Mix 6μL of PCR product with 1μL of Loading buffer,and use 1.5%agarose gel electrophoresis to separate the products,and then screen out the optimal dosage of each factor according to the electrophoresis bands to choose the optimal reaction condition.4.Establishment and evaluation of MPCR-CE systemModify the FAM fluorophore on the 3’end of the target bacteria-specific primers.After PCR amplification is completed,the product DNA fragments are separated by capillary electrophoresis with ABI 3500-Dx DNA Analyzer,and the results are analyzed using Genemapper V5.0（Applied Biosystems）.To evaluate the specificity of the established MPCR-CE system,four primers were added according to the optimized conditions,each target bacteria template DNA was added separately,and four non-target bacteria were selected as specific controls,and dd H₂O was used as a negative control.Use optimized reaction conditions for detection,and analyze the presence or absence of cross-reaction mismatches between primers and templates based on the results of amplification products to verify the specificity of the system for detection of target bacteria.Subsequently,in order to evaluate the sensitivity of the MPCR-CE system,DNA of the four target bacteria were extracted and diluted with dd H₂O successively 10 times,so that the concentration of the template DNA of the four bacteria was in the same order of magnitude(10²-10^-8ng/μL).Use capillary electrophoresis and 1.5%agarose gel electrophoresis to detect the amplification results separately,and compare the sensitivity and specificity of the two signal detection methods.5.Detection and evaluation of simulated samples based on Mag-Cds-MPCR-CE technologyThe four kinds of food-borne pathogenic bacteria were mixed and then diluted by 10 times（10⁵-10⁰CFU/mL）,and added to the sterile milk sample to prepare a simulated sample.Use Mag-Cds to magnetically separate and enrich the four pathogens,and use the optimized MPCR-CE system for template DNA amplification and amplification product detection,and determine the detection sensitivity of Mag-Cds-MPCR-CE system.The four pathogens were randomly mixed in two or three and four common Non-target food-borne pathogens such as Vibrio parahaemolyticus,Enterococcus faecalis,Shigella baumannii and Klebsiella pneumoniae were used to verify the specificity of the Mag-Cds-MPCR-CE system.Chicken sausages pears and other ready-to-eat foods were used to make simulated samples to test whether the system can be used in different food sample matrices.Result:1.The result of characterization of Mag-CdsObserving the morphology and particle size of the Fe₃O₄MNPs and Mag-Cds prepared by transmission electron microscope and fourier infrared spectrometer,it can be seen that most of the Fe₃O₄MNPs prepared are spherical or nearly spherical with uniform particle size,with a slightly rough surface,good dispersion,and a diameter of approximately 20 nm.At the same time,the transmission electron microscope image shows that Mag-Cds particles are uniformly dispersed.Because the outer layer is coated with carbonized chitosan,its particle size is slightly larger than that of bare Fe₃O₄MNPs,with a diameter of about 20 nm.The fourier infrared spectrometer spectrum showed that the two kinds of nanoparticles have their own characteristic peaks,indicating that carbon has been successfully coated on the surface of Fe₃O₄MNPs.The two kinds of nanoparticles before and after modification are potentially superparamagnetic and can be manipulated by an external magnetic field.2.The result of Mag-Cds determination of magnetic enrichment efficiency for four food-borne pathogensThe capture efficiency of S.aureus and E.coli O157:H7 reaches 85.5%and87.8%,respectively,which indicates that most of the S.aureus and E.coli O157:H7 has been captured and enriched by Mag-Cds.Therefore,the dosage of Mag-Cds in this experiment was selected as 6 mg/mL.Under this dosage,the enrichment efficiency of Mag-Cds for S.aureus is 86.5%,the enrichment efficiency for L.monocytogenes is 94.3%,and the enrichment efficiency for S.typhimurium is 90.9%,and it is 90.9%for the large intestine.The enrichment efficiency of Bacillus O157:H7 was 89.9%.3.The result of establishment and evaluation of MPCR-CE systemAccording to the screening,the final selection of L.monocytogenes hemolysin gene（hlyA）,S.aureus heat-resistant nuclease gene（nuc）,S.typhimurium adsorption and invasion epithelial cell surface protein coding gene（inv A）and E.O157:H7antigen-specific gene（rfbE）as the target gene.After screening and identifying the primers and ensuring that the amplification can proceed smoothly,this study optimized the reaction conditions in the multiplex PCR reaction system,and finally determined the best reaction system.The reaction system was a total volume of 50μL.Includes 5μL 10×PCR buffer,7μL Mg²⁺（25 mM）,5μL d NTPs,0.6μL（5U/μL）DNA Polymerase enzyme,2.4μL（10μmol/L）of L.monocytogenes primer,2.0μL of S.aureus primer（10μmol/L）,2.0μL of E.coli O157:H7 primer（10μmol/L）,1.6μL of S.typhimurium primer（10μmol/L）.Each template DNA 2μL,and sterilized water is made up to 50μL.The optimal reaction conditions are:pre-denaturation at 94°C for 5 min;denaturation at 94°C for 30 seconds,annealing at 55°C for 30 s,renaturation at 72°C for 1 min,30 cycles,and finally extension at72°C for 10 min.4.The result of Establishment and evaluation of MPCR-CE systemThe multiple PCR products were subjected to capillary electrophoresis and agarose gel electrophoresis,respectively.The capillary electrophoresis results showed that under the optimized reaction conditions,the expected specific amplification peaks appeared in each target bacteria.The size of L.monocytogenes was 439 bp and the fragment of S.aureus was 230 bp,the fragment size of E.coli O157:H7 is 151 bp,and the size of S.typhimurium is 305 bp.The fluorescence value of each product is high and there is no crossover area,and the 4 kinds of non-target strains have no specific amplification peaks.The results of agarose gel electrophoresis showed that the positive control and each target bacteria showed expected electrophoretic bands,while the negative control non-target bacteria did not show amplified bands,indicating that the method has good specificity.The results of agarose gel electrophoresis showed that the detection limits for the four pathogenic bacteria were all 10^-3ng/μL.As can be seen from the result graph of the capillary electrophoresis analysis software that the detection limit for S.aureus can reach 10^-7ng/μL,the detection limit for E.coli O157:H7 can reach 10^-6ng/μL,and the detection limit for S.typhimurium and L.monocytogenes can reach 10^-5ng/μL.5.The result of testing and evaluation simulated samples based on Mag-Cds-MPCR-CE technologyThe test results of artificially contaminated food samples show that the target genes of the four pathogens selected in the experiment can be successfully amplified,the amplification results are consistent with the expected target fragment size,and neither the negative control nor the non-target bacteria showed specific amplification.Random combination of the four bacteria in the simulated food sample can amplify their respective target fragments.The detection limit of the system for the four pathogens can reach 10¹CFU/mL.In addition,the four pathogens can be detected smoothly in different sample matrices.The peak values of capillary electrophoresis in pear juice and chicken intestine samples are higher than milk and biscuit samples.The test results of simulated samples show that the established multiplex PCR detection method is fast,simple,specific,and sensitive,and is suitable for different food sample matrices,and has high practical application value.Conclusion:1.This research integrates magnetic separation technology,multiple PCR technology,and capillary electrophoresis technology to construct Qualcomm for S.typhimurium,S.aureus,L.monocytogenes and E.coli O157:H7 in food samples.High-volume,fast and accurate Mag-Cds-MPCR-CE detection system.2.The Mag-Cds-MPCR-CE reaction system can complete the simultaneous detection of four food-borne pathogens within 150 min.The detection limit for genomic DNA of Mag-Cds-MPCR-CE can reach 10^-5ng/μL～10^-7ng/μL,while it for simulated food samples were 10¹CFU/mL with excellent specificity.It has high sensitivity and good specificity and can meet the requirements of food safety testing.