Establishment And Application Of Rapid Molecular Detection Technology For Salmonella In Ready-to-Eat Fruits And Vegetables

The ready-to-eat fruits and vegetables with benefits for health and unique flavors have become more and more attractive to consumers.Over the past decade,consumption of freshcut fruits and vegetables has increased significantly,but eating in raw has increased the frequency of outbreaks of foodborne pathogens illness.According to statistics,Salmonella is the primary pathogen in bacterial food poisoning of mainland China,and the outbreak of pathogenic bacteria caused by Salmonella contamination is recognized as a food safety problem worldwide.Countries such as China,the WHO and the European Union have listed Salmonella as a compulsory detection item for food,and sanitary standards all require Salmonella must not be detected in any product.Therefore,it is critical to establish a rapid and sensitive method for Salmonella contaminated in ready-to-eat fruits and vegetables to minimize the risk of infection from the source of the pollution.In this study,we developed a selective enrichment broth for Salmonella,and based the laboratory-constructed Salmonella loop-mediated isothermal amplification(LAMP)method to assemble an LAMP wet kit and an one-step LAMP lyophilized kit suitable for actual detection.The LAMP kits achieved qualitative analysis of Salmonella in ready-to-eat fruit and vegetable samples.Meanwhile,the highly specific sequence of S.Enteritidis Gene ID:1828390769 and Salmonella spp.Gene ID:3335471,screened by this laboratory,was used to establish S.Enteritidis and Salmonella spp.real-time quantitative PCR detection systems,which could quantitative analysis of Salmonella in samples.The main results are reproduced below.A novel selective enrichment broth(Selective Luria-Bertani Broth,SLB)combined with a loop-mediated isothermal amplification(LAMP)kit was developed for inspection of Salmonella in ready-to-eat fruits and vegetables.SLB was formulated by Tween 80,sodium deoxycholate,mannitol as promoter,sodium pyruvate,lithium chloride,glycine as inhibitor incorporation into the Luria-Bertani(LB).The recovery of acid-injured Salmonella in SLB was superior to Selenite Cystine Broth(SC)(p<0.05).Additionally,the growth speed of Salmonella in SLB was exceeding among LB and SC,and growth inhibition of non-target bacteria without compromising Salmonella.Fruits and vegetables naturally polluted by microbiotas were artificially inoculated with Salmonella.After incubation in SLB(10 hours)and Buffered Peptone Water(BPW)-SC(34 hours),the LAMP kit had a high detection rate(100%).Detection by a streak plate method,BPW-SC was lower than SLB.SLB has good solubility,reduced incubation times,the combination of SLB and LAMP could enhance the accuracy of test results.The purpose of this study was to assemble two types of loop-mediated isothermal amplification(LAMP)kits that have the ability to visually detect Salmonella in ready-to-eat fruits and vegetables.The reaction results were obtained within 20-40 minutes after addition of DNA and can be discerned by the naked eye or an amplification plot.The stability of the LAMP wet kit was manifested in multiple freezing and thawing cycles,and the one-step LAMP lyophilized kit was further evolved to allow ambient temperature transport for deployment in resource-limited settings.The cost-effective wet kit had the ability to detect minimum amounts of 1.8 CFU/mL Salmonella DNA without enrichment,while the sensitivity of the one-step LAMP lyophilized kit was only 9.8×103 CFU/mL.They both have good anti-interference,as they were both able to detect 2.1 ×102 CFU/mL Salmonella mixed with 106 CFU/mL four non-Salmonella mixture.Moreover,cucumber and lettuce that were contaminated with an initial inoculation of 1.7 CFU of Salmonella/10 g showed detection within a reaction time of 30 minutes after 10 hours enrichment.The present research setup is a convenient and practical kit for Salmonella rapid detection that has good application prospects in food safety monitoring.The visualization kit has low detection limits,high accuracy,simple and fast operation,good specificity,can meet the detection requirements,high commercial feasibility.Primers were designed based on the highly specific sequence of S.Enteritidis Gene ID:1828390769 and Salmonella spp.Gene ID:3335471.Through the optimization procedure of amplification system and program,and screened the fluorescent dyes and concentration,two real-time quantitative PCR detection systems,contained DNA Green saturated fluorescent dye for detection S.Enteritidis and included Ly Green saturated fluorescent dye for detection Salmonella spp.,were constructed.Furthermore,the specificity of the amplification system had increased with the addition of graphene quantum dots.The R2 of the standard curve was reached at 0.999(S.Enteritidis)and 0.998(Salmonella spp.),respectively.And the sensitivity of the two amplification systems was evaluated from three aspects:genomic DNA(1.36fg/pL,1.36fg/?L);bacterial cell without enrichment(2.49 CFU/mL,2.49 CFU/mL);plasmid containing the Gene ID:1828390769(11.6 copies/?L)and plasmid containing the Gene ID:3335471(1.08 copies/?L).At lower genomic DNA and plasmid concentrations,the SD of the Ct values of the two amplification system was<0.5,within the acceptable range,and the repeatability was good.Detection results of Salmonella artificially contaminated fruit and vegetable showed that the traditional culture method has a lower detection rate than the molecular detection method,and the culture time of the SLB was short,and the detection rate was higher than that of the BPW-SC.The lettuce samples were detected by LAMP wet kit and Salmonella real-time quantitative PCR method after 10 hours of SLB bacterial growth.The detection rate of Salmonella was 2/41,which was consistent with the experimental results of the biochemical identification kit.S.Enteritidis was not detected by real-time quantitative PCR.However,after the national standard method of cultivation(culturing time greater than 34h),the detection results of the three methods was inconsistent.The two detection system has high sensitivity and specificity,and suitable for the detection of Salmonella in ready-to-eat fruits and vegetables.