Detection Method Of Viable Cronobacter Sakazakii

Food safety is a global public health safety issue.It has always been the focus of social concern for relating to the vital interests of everyone,among which,food safety issues caused by foodborne pathogens are the focus of most concern recently.Foodborne pathogens are very harmful and have a great impact on food contamination,so it is of great significance to establish fast,convenient and sensitive detection methods for foodborne pathogens to prevent food safety issues and protect human health.Cronobacter sakazakii is a foodborne opportunistic pathogen that causes a range of diseases,including sepsis,necrotizing colitis and neonatal meningitis.It can be found in a variety of foods such as milk,vegetables,meat and tea.However,infant formula is the main source of contamination.C.sakazakii has been officially identified as a class A pathogen by FAO-WHO because of its great potential risk to newborns.In this paper,based on the specific sequences and special structure of C.sakazakii,four kinds of rapid detection methods for viable C.sakazakii were established as follows:1)An electrochemical detection method for viable C.sakazakii was established based on RT-PCR and DNAzyme.In this study,the specific primers for theα-1,6-glucosidase gene of C.sakazakii were firstly designed,and a large number of DNA products containing G-quadruplex were amplified by RT-PCR through primer modification.In the presence of hemin,G-quadruplex/hemin DNAzymes with peroxidase activity were formed to catalyze the oxidation of substrate TMB to generate oxides with electrochemical activity.The enzyme-catalyzed electrochemical signals through screen-printed carbon electrode（SPCE）were obtained by chronoamperometry,the catalytic products were characterized,and the number of viable C.sakazakii in the samples was quantitatively analyzed by the relationship between the value of current signal and the content of target gene in the sample.By evaluating the specificity and sensitivity of the detection method,the results showed that the method had high specificity and the detection limit was 5.01×10² CFU/m L.The electrochemical biosensor detection method was simple and convenient to operate,had stable current signal,and required only trace amounts of detection system,which was conducive to popularization and application.2)A rapid visual detection method for viable C.sakazakii was established based on LAMP-PMA.As a dye with high affinity to DNA,PMA could enter the dead bacterial cells with incomplete cell walls or cell membranes,and then bind with the DNA.LAMP amplification combined with PMA could effectively inhibit DNA amplification in dead bacteria cells,thus to realize the detection of viable bacteria.LAMP primers were designed according to the specific sequences of C.sakazakii omp A gene,and the PMA-treated DNA of bacteria was used as the detection target.HNB was used as a metal ion indicator to transform the molecular amplification to optical signals,so as to realize rapid visual detection of viable C.sakazakii by naked-eye observation.After the feasibility test,the experimental conditions were optimized,and the specificity and sensitivity of the detection method were evaluated under the optimized conditions.The results showed that the visual detection method of viable C.sakazakii based on LAMP-PMA had the advantages of simple experimental equipment,simple operation,good specificity,and the sensitivity was3)A rapid visual detection method for viable C.sakazakii was established based on RT-PSR.As a novel isothermal amplification method,polymerase spiral reaction（PSR）could denature DNA without heating and helicase,and complete nucleic acid amplification under isothermal conditions.Based on the isothermal amplification characteristics of PSR,RT-PSR primers were designed with the RNA of C.sakazakii as the detection target,and HNB was added into the amplification reaction as an indicator of Mg²⁺concentration in the system.Through the color change of the system,rapid visual detection of viable C.sakazakii was realized.Firstly,the experimental conditions were optimized,and then the specificity and sensitivity of the visual detection method were evaluated under the optimal experimental conditions.The results showed that the detection strategy had the advantages of short time,low cost and high specificity,and the sensitivity was 1.2×10 CFU/m L.4)A rapid visual detection for viable C.sakazakii was established based on nucleic acid aptamer and DNAzyme.The aptamer of viable C.sakazakii was synthesized and the probes containing fractional complementary sequences of aptamer and two split G-quadruplex halves were designed and synthesized according to the aptamer.The detection method directly used viable C.sakazakii as the detection target.While the target did not exist,the two probes were complementary to the aptamer,and the G-quadruplex sequences of the two probes were bound to constitute the G-quadruplex structures,which formed the G-quadruplex/hemin DNAzyme with peroxidase activity in the presence of hemin.The DNAzyme catalyzed the substrate ABTS to generate optical signals with H₂O₂,finally realized to directly identify the presence of viable C.sakazakii in the sample by naked eye,and the sensitivity of this detection method was 1.2 CFU/m L.Through further evaluation of the specificity of this method,the results showed that the method had a high specificity.This study showed that the strategy of visual detection for viable C.sakazakii based on aptamer and DNAzyme was a stable,enzyme-free,label-free,low-cost detection method with high specificity and high sensitivity.