Research On Sample Pretreatment Method For Highly Sensitive Detection Of Foodborne Pathogenic Bacteria Based On Rti-LAMP

Food safety caused by foodborne pathogens is an important public health problem affecting people’s health.Escherichia coli O157:H7 and Salmonella are often contaminated foodborne pathogens with low infection dose and strong pathogenicity.The establishment of rapid and sensitive detection methods of E.coli O157:H7 and Salmonella in food is very important to ensure food safety.In the present study,a method for rapid detection of E.coli O157:H7 in milk was established by combining Rti-LAMP with immunomagnetic separation（IMS）.The specific immunomagnetic beads（IMBs）of bacteria were prepared by coupling carboxylated nano-magnetic beads with E.coli O157:H7 polyclonal antibody.Taking the 1m L bacterial sample as the detection object,the immunomagnetic beads were used to capture（IMS）;by optimizing the proportion of antibody magnetic beads,the dosage of immunomagnetic beads,the reaction time and the magnetic separation time.The results are as follows:under the conditions of 50μL of E.coli O157:H7 immunomagnetic beads,reaction 30 min and magnetic separation 1 min,the efficiency of capturing E.coli O157:H7 is more than 90%.Under the optimized conditions,the samples E.coli O157:H7 cultured in 1 m L were captured and recovered by IMS method.After EMA treatment,the DNA was extracted as template,and the established Rti-LAMP method was used for amplification and detection.The results showed that the sensitivity of living E.coli O157:H7 in pure culture was 2.7CFU/m L.The contaminated milk samples were artificially simulated with E.coli O157:H7and live-dead mixed E.coli O157:H7 respectively,and the 1m L samples were detected by IMS-EMA-Rti-LAMP.The results showed that the established IMS-EMA-Rti-LAMP method could quantitatively detect the live E.coli O157 H7 in milk in the presence of dead bacteria,and the whole detection process was only about 2.5 hours.The method was rapid,sensitive,specific and without enrichment for the detection of target pathogenic bacteria in milk.Viable but non-culturable（VBNC）state is a special survival strategy of some bacteria under environmental stress.To establish the induction law of Salmonella VBNC state,Salmonella with high and low concentrations（1.3×10⁶,1.3×10⁴CFU/m L）were frozen and thawed for several times,and the number of living bacteria after each freeze-thaw was detected by plate counting method,direct fluorescence counting（DEM）method and EMA-Rti-LAMP method.The results showed that there was no significant difference in the number of living bacteria obtained by EMA-Rti-LAMP and DEM methods,and both were higher than the plate count results,which proved that some Salmonella were transformed into VBNC state during repeated freezing and thawing,and EMA-Rti-LAMP method could detect viable Salmonella including VBNC.In addition,after 4 times of freezing and thawing,the low concentration of Salmonella could completely disappear,while the high concentration of salmonella still had 2.7 Lg CFU/m L culturable bacteria,indicating that the high concentration of Salmonella had higher tolerance to freeze-thaw.To study the VBNC changes of Salmonella under cold storage and freezing conditions,Salmonella with high and low concentrations（1.3×10⁶ and 1.3×10⁴CFU/m L）were stored in saline at 4℃and-20℃,respectively.The changes of live bacteria and culturable bacteria with time were investigated.Results the number of viable Salmonella in high concentration Salmonella was basically stable at 4℃,while the number of culturable bacteria decreased slowly.Similarly,the number of viable bacteria in the low concentration group preserved at4℃was relatively stable,and the number of viable Salmonella decreased slowly until it disappeared completely at 110 d.The results at-20℃were slightly different,the number of viable bacteria decreased rapidly in the low concentration group,and there was no living bacteria on the 10th day of preservation,while in the high concentration group,the number of living bacteria decreased about 2 Lg CFU/m L in the first 60 days,the number of culturable bacteria disappeared on the 110th day,and Salmonella 4.31 Lg CFU/m L still survived in VBNC state.To establish a method for rapid detection of Salmonella in VBNC state in food,Salmonella induced to VBNC state was artificially inoculated into contaminated chicken,the EMA-Rti-LAMP combined with bentonite-coated activated carbon（BCAC）treatment could detect as low as 35 CFU/g VBNC Salmonella derived from contaminated chicken,and the entire assay completed in 5 h.Furthermore,four identical samples were Salmonella positive from 24 retail frozen chicken samples detected by plate culture（GB4789.4-2016）,BCAC-Rti-LAMP,and BCAC-EMA-Rti-LAMP.The BCAC-EMA-Rti-LAMP had one more sample for Salmonella positive than that of plate culture,but less two samples than that of BCAC-Rti-LAMP.Noticeably,the BCAC-EMA-Rti-LAMP had much more accuracy as plate counting than that of BCAC-Rti-LAMP in detection of viable Salmonella derived from chicken.These results suggested that the BCAC-EMA-Rti-LAMP assay could be a rapid and sensitive method for detection of viable Salmonella including VBNC cells in chicken without enrichment.Reaction inhibitors that affect nucleic acid amplification methods such as PCR and LAMP are generally considered to have two categories:water-soluble inhibitors and lipids.After BCAC and IMS treatment,the interference level of water-soluble inhibitors is reduced to an acceptable level,but there is a lack of effective treatment methods for oil-rich food samples.In this study,using natural potato starch as raw material,a kind of enzymatic hydrolyzed porous starch（HPS）was prepared by ultrasonic treatment andα-amylase treatment for 30 minutes.Taking the bacterial recovery efficiency of 10g artificially simulated oil content of about 15%as an index,the addition amount ofα-amylase and the reaction time of enzyme were optimized.The results showed that the amount of enzyme was 315 U/g,and the action time was 8 h.The recovery rate of bacteria was 82.4±2.1%.Then the adsorption kinetics of animal oil and vegetable oil by HPS was studied.The adsorption of animal oil and vegetable oil by HPS conformed to the quasi-second-order adsorption kinetic equation.It was inferred that the adsorption was mainly the chemical adsorption of functional group sharing or electron exchange.The characteristics of HPS were studied by scanning electron microscope,surface enhanced Raman spectroscopy and zero charge point characterization.Ultrasonic treatment andα-amylase pore formation marks were observed on the surface of HPS under SEM.Raman spectra proved the change of starch structure.p H_pzc=6.78 proved that HPS and bacteria were negatively charged,and charge repulsion was helpful for bacteria recovery in neutral buffer environment.To verify the actual detection effect of HPS in oil-rich food,artificial simulation of different concentrations of Salmonella minced beef samples,HPS treatment combined with BCAC-Rti-LAMP to detect simulated contaminated samples.The results showed that after HPS treatment,the sensitivity of Salmonella in ground beef samples was10 CFU/g,which was 10 times higher than that of untreated group.It shows that HPS can effectively recover Salmonella from food samples rich in lipids and improve the detection sensitivity.