Study Of Novel Rapid Detection Methods Of Escherichia Coli O157:H7 And Campylobacter Jejuni In Food

Food safety is an increasingly prominent social problem,which concerns the vital interests of millions of Chinese people.It is imperative to pay attention to food safety.Foodborne pathogen,which is highly hazardous and widespread,is one of the most common food contaminations.Therefore,it is very important to establish the convenient,rapid and sensitive detection methods of foodborne pathogen to prevent food safety problems and protect human health.This paper introduces several rapid detection methods of E.coli O157:H7 and Campylobacter jejuni that are as two common foodborne pathogens.(1)Establishment of identification of living/dead bacteria based on molecularamplification principlePropidiummonoazide(PMA)was incubated with bacterial cells.Under normal circumstances,PMA could not pass through the intact cell membrane.But in the case of death,there was degradation or structural damage on the cell membrane,so PMA would selectively enter the inside of the dead cell to bind to its DNA.When bacterial samples treated with PMA are exposed to special light for a certain period of time,the PMA will embed in the double strand of the DNA molecule,resulting in permanent modification of the DNA molecule.In addition,covalent DNA modification can inhibit the activity of polymerase and thus block the polymerase chain reaction(PCR)of DNA molecules.Therefore,DNA from non-living or membrane-damaged cells cannot be amplified by PCR.After the optimization of experimental conditions,when the PMA with a final concentration of 5 g/mL was incubated with the bacterial sample for 5 minutes and exposed under 500 W halogen lamp for 5 minutes,the PMA could exert the maximum inhibitory effect on the PCR amplification of DNA.The method is used to judge the dead or living state of bacteria and pave the way for further selection and establishment of detection methods for bacteria.(2)Study of the detection of E.coli O157:H7 based on magnetic enriched lateralchromatographic stripBased on the immune recognition between antigen-antibody,the gold nanoparticles(GNPs)marked monoclonal antibody as recognition probe combined with E.coli O157:H7.Then they were captured by another anti-E.coli O157:H7 antibody immobilized on the test line(T line)and formed the sandwich structure.Finally,GNPs were fixed on the T line to make T line visible.The GNPs marked the antibodies were captured by secondary antibodies fixed on the control line(C line)and made C line visible.The concentration of E.coli O157:H7 in the system under test was quantitatively analyzed by observing the color intensity of T line by naked eyes.On this basis,the magnetic nanoparticles(MNPs)labeled with anti-E.coli O157:H7 antibody were added to the sample for magnetic enrichment,and then applied to the colloidal gold immunochromatographic strip for detection.Under the optimal experimental conditions,the detection sensitivity of the traditional chromatography strip could reach to 4.1×10~4cfu/mL.In contrast,the magnetic enhanced strip could increase the detection sensitivity by 100 to 1000 times,and 10~1 cfu/mL of E.coli O157:H7 could be detected by naked eyes with good detection linearity and specificity.(3)Study of the detection of virulence gene of E.coli O157:H7 based on PCRVirulence gene is as a specific fragment in bacterial DNA.Each bacterium has its unique virulence gene,so virulence gene can be detected at the molecular level to detect specific bacteria.Based on this,we first extracted the DNA of E.coli O157:H7 using MNPs,and then specific virulence gene(rfb)was amplified by PCR with specific primers.Finally,the PCR products were characterized by agarose gel electrophoresis,and the concentration of E.coli O157:H7 in the system under test was preliminarily quantitatively analyzed by observing the strength of electrophoresis strips of target products by the naked eyes.After the optimization of experimental conditions,PCR amplification could detect 10~2 cfu/mL of E.coli O157:H7 with good detection specificity.(4)Study of the detection of virulence gene of E.coli O157:H7 based on lateralchromatographic fluorescence stripBased on the immune recognition between antigen and antibody and the specific recognition between streptavidin(SAV)and biotin(biotin),the 5'end of both forward and reverse primers were respectively modified biotin and isothiocyanate fluorescence(FITC),and then PCR was used for amplification.And the products labeled with biotin and FITC at both ends were obtained.PCR products were firstly combined with anti-FITC antibody labeled on the surface of fluorescence microspheres(FMs)and then captured by SAV fixed on the T line.T line was visible under ultraviolet light.FMs labeled with anti-FITC antibody were captured by the secondary antibody fixed on C line,and the C line was visible under ultraviolet light.The concentration of E.coli O157:H7 in the system under test can be preliminarily quantitatively analyzed by observing the fluorescence intensity of T line with the naked eyes.Under optimal experimental conditions,3 cfu/mL and 3×10~1 cfu/mL of E.coli O157:H7 can be detected in the standard sample and the real sample respectively.Compared with the PCR results,this method can improve the detection sensitivity by two orders of magnitude and has good detection linearity and specificity.(5)Study of the detection of Campylobacter jejuni by sandwich colorimetricprotocol based on heterogeneous recognition systemThe aptamer against Campylobacter jejuni was screened based on the classical whole-bacteria SELEX method,and we used the antibody and the selected aptamer against Campylobacter jejuni to construct an antibody-aptamer sandwich colorimetric protocol based on heterogeneous recognition system to detect Campylobacter jejuni.By forming a sandwich structure,horseradish peroxidase(HRP)was fixed in the micropore,and finally the TMB substrates were added for color development.Visual observation of the color of the liquid in the micropore can preliminarily and quantitatively analyze the concentration of Campylobacter jejuni in the system under test.After the termination with concentrated sulfuric acid,the optical strength value of the liquid in the microporous was measured by a microplate reader,which could be used to quantitatively analyze the concentration of Campylobacter jejuni in the system under test.After screening and analysis,we select the sequence A1(5'-CTGCGATCAAGTTACGCACCTCGCCATGTT CCCCGCCCGGCATGTGTTATGCCCCTGTG-3')as the aptamer against Campylobacter jejuni.Under optimal experimental conditions,sandwich colorimetric protocol based on heterogeneous recognition system can detect Campylobacter jejuni with 13 cfu/mL,and it has good detection linearity and specificity.This further indicates that the aptamer screened against Campylobacter jejuni in this study has good properties.