The Development Of Visual Methods For Salmonella Detection Based On Hybridization Chain Reaction

As the main part of international trade,food trade expands export exchange,solves the contradictory between grain supply and demand and at the same time brings an increase in morbidity and mortality of foodborne disease.In our country,microbial pathogen is a major cause of foodborne illness,making up 30%~40%.And the bacteria accounted for 81.5% of microbial pathogens.Therefore foodborne illness,especially pollution caused by bacteria,is the top issue of food safety in our country.As a big country in food production and consumption,to establish the rapid and accurate foodborne pathogen detection technology is very important for the food quality control and people’s health assurance.There are three main categories of detection methods for food-borne bacteria: plate-culture methods,immunochemical detecting methods and molecular biology of gene methods.Traditional culture method doesn’t need complex experimental instruments and the detection results are reliable,but the whole process lasts too long(5~7 days)and takes time and energy.Despite of high specificity and sensitivity,immunological detection methods,such as ELISA,target antigens or antibodies.That is to say,these detection methods need protein reagent,such as monoclonal antibody,and also require harsh conditions.New immunofluorescence techniques are also selective to reagents and instruments.Because the nucleic acid is trace in living organisms,amplification processes are necessary for most modern molecular biology techniques,such as polymerase chain reaction PCR,real time quantitative PCR.Nucleotide amplification based on temperature cycle requires precious equipments,is not suitable for small laboratories as well as the backward area.Hybridization chain reaction(HCR)is a nucleic acid amplification reaction,which is enzyme-free and performed at room temperature conditions.Since no enzyme involved in the reaction and the low demand for reaction condition,HCR in combination with electrochemical and fluorescence technique has been used in detection of different targets such as nucleic acid,protein and pathogen in recent years.The detection results of visual methods could be unambiguously visualized by the naked eye under the visible light or ultraviolet light.Comparable to other assays,visual methods are more commonly applied because of their low cost.The purpose of this study is to establish a quick and low-cost detection method for food-borne pathogens,which takes HCR as amplification method.We take salmonella as target model.First,we find the specific sequence in 16 S rRNA of Salmonella enteritidis by the Basic Local Alignment Search Tool of NCBI and design the specific capture probe and detect probe,then we combine HCR with two detection platform: lateral flow biosensor and colorimetric detection based on microplate to establish two visual detection methods.For the lateral flow biosensor,we first need to prepare the colloidal gold immune chromatography test paper: streptavidin coated AuNPs,anti-FITC mAb coated test lines and biotin coated control lines.Then synthetic sequence are used for the establishment of model and the optimal experiment conditions were confirmed: the best concentration ratio of start probe/hairpin probe was 1:5,the ration of modified hairpin probes/hairpin probes was 9:1,the optimal concentration of capture probe is 0.3μM.This method has excellent specificity that can differentiate target from its similar sequences.And its LOD for synthetic sequence is 1.76 pM,which is magnified 176-fold,the LOD for bacteria solution is 3*103CFU mL-1.After acquisition of RNA sample,the whole detection process can be completed in 30 min,and the final result can be observed.In microplate and color experiment,streptavidin coated plates were set for fixing capture probe with specific recognition ability.The existence of the target sequence enables sandwich structure: grab probe/target sequence/HCR to form,which were fixed to the plate.Due to the FITC tag of HCR product,horseradish peroxidase labeled fluorescent antibody anti-FITC – HRP was also fixed.The existence of HRP not only catalyzed chromogenic reaction,but also achieved double signal amplification.We also optimize the condition of experiments: the best concentration of capture probe is 0.5μM,the best incubation for capture probe is 2 hours,the optimal incubation time for anti-FITC-HRP is 2 h.The capture probe is enough to distinguish between the target and one-base different sequence.The LOD for synthetic sequence is 32.6 fM.In real sample detection,it can not only distinguish between target bacteria group and no-target bacteria group(the signal ratio of non-target groups/ target group was not higher than 22%),but also has the LOD of 52.1 CFU mL-1.The above results indicated that the two visual methods had strong specificity and high sensitivity.Relative to methods that use technology such as electrochemical and fluorescence,the two methods introduced in this article is not the best in terms of detection sensitivity,but both of their results can be judged by the naked eye,which doesn’t need high-end instrument and the whole experiment process is simple,which doesn’t need professional operation.Therefore they are suitable for remote and resource-poor areas,and can be used as POCT diagnostic reagents for further development and application.