Study And Application Of Accurate Test Method For Campylobacter Jejuni And Salmonella In Chicken

The proportion of food-borne diseases caused by pathogens has increased in recent years and has become the focus of global public health.It is highly concerned by the World Health Organization and many countries.Now China is highly concerned about people’s health.Food safety guarantee has become an important task for governments and food supervision authorities.Campylobacter jejuni and Salmonella,the food-borne zoonotic pathogens capable of causing various diseases in human and animals,are amongst the most important pathogenic factor of bacterial diarrhea.They are two important organisms in food-borne pathogens detection and control for food safety surveillance and management.Poultry is the primary host animal for the two pathogenic bacteria,and they spread through food chain from farm to folk.Chicken and its products is the most important food carrier of Campylobacter jejuni and Salmonella.It is urgent to issue food safety warning to ensure the public health.Therefore,the rapid and accurate detection and identification system for Campylobacter jejuni and Salmonella in chicken meat has become one the most urgent research concerns.This study mainly includes the following five sections:1.Construction of plasmid positive control of Campylobacter jejuni and Salmonella and accurate quantification with digital PCRIn this chapter,the plasmid positive control of Campylobacter jejuni hipO gene was constructed.The hipO gene of C.jejuni was inserted in a plasmid and used as positive control for ddPCR detection method,the concentration of hipO gene was 6.18×108 copies/μL.The invA gene recombinant plasmid for Salmonella was constructed as positive control,and the ddPCR detection method based on invA gene was developed to determine the quantity of plasmid positive control,the confirmed concentration was 8.53×107 copies/μL.2.Optimization and preliminary application of Real-time PCR of Campylobacter jejuni and SalmonellaIn this chapter,accurate quantitative Campylobacter jejuni and Salmonella plasmid positive controls are seprately used to evaluate and optimize the key influencing factors of the real-time PCR detection method.The Life 7500 real-time PCR instrument was used to evaluate the reaction master mix.At low concentrations(10-6 and 10-7),MeiKang PCR mastermix showed higher amplification efficiency and stable results.The CV value of the C.jejuni plasmid positive control on the MK-V280 platform was 1.27%for the sensitivity test,and t for the repeatability test at medium and low concentrations the CV values were 0.98%and 0.86%,respectively.The specificity was consistent.In contrast,the CV value of the above sensitivity test was 1.92%at high concentration on Life 7500 platform,and at the medium and low concentration they were 0.71%and 0.86%,respectively.the specificity was consistent as well.The Salmonella plasmid positive control had a CV value of 3.58%in the sensitivity test on the MK-V280 platform,and CV value of 2.0%and 1.31%in the repeatability test at medium and low concentrations,respectively,and the specificity was consistent.The sensitivity CV value of the test was 1.05%on the Life 7500 platform,and the CV values of the repeatability test at the middle and low concentrations were 1.04%and 3.08%,respectively,and the specificity was consistent.500 chicken samples were test by the optimized real-time PCR detection method,it was shown that 36 samples was Campylobacter jejuni positive of PCR,32 of which were consistent with biochemical identification results.53 samples showed Salmonella positive of PCR,and the biochemical identification results were completely consistent.3.Sequencing and character analysis for hipO gene of Campylobacter jejuniIn this chapter,hipO gene of 8 Campylobacter jejuni strains were cloned and sequenced,and sequence comparison and characterization of 17 Campylobacter jejuni strains were performed.In the gene sequence of the four isolates with negative hippuricase hydrolysis reaction and four positive strains,the 98th base is from G to A,the 372th base is from C to T,and the 372th base is from T to C,the 576th base from A to G,the 603th base from A to G,the 640th base from A to G,the 648th base from A to G,the 969th base from C to T,the 1002th base is from A to G.The corresponding amino acid sequence at position 33 is from C(cysteine)to Y(tyrosine),and the amino acid at position 324 is from H(histidine)to Y(tyrosine).The gene homology of A110725,A130506,H130311,and D130621,which are negative for the reaction of the uric acid hydrolysis,is 98.5%-99.6%,and the protein homology is 98.2%-99.2%.4.Improvement and preliminary application of MALDI-TOF MS identification database for Campylobacter jejuniThe research in this chapter proves that the choice of matrix fluid affects the peak intensity of Campylobacter detection,and confirmed that the trifluoroacetic acid 0.5%(v/v),and the peak intensity is the highest,which meets the detection requirements.The MALDI-TOF MS identification results of 13 Campylobacter standard strains were consistent,and the bacteria of the same species were classified as one branch,and the results were integrated into the VITEK MS database.Key characteristic peaks of m/z 6157,7034,8153,8460,9552 and 10276 were identified for Campylobacter jejuni.The optimized identification database was used for the identification of 20 Campylobacter jejuni isolates,which were consistent with the sequencing results.The confidence rate of Campylobacterjejuni identification was higher than 99%.5.Study on the colorimetric detection method of Salmonella typhimurium based on aptamer functionalized nano-gold probeIn this chapter,a colorimetric detection method for Salmonella typhimurium based on an aptamer functionalized nano-gold probe is established.The biotinylated Salmonella typhimurium aptamer was immobilized on avidin-coated microplate wells,and the target bacteria and avidin-catalase were sequentially added to the wells of the microplate.Hydrogen peroxide and freshly prepared chloroauric acid were added,and the absorbance of the reaction product was measured by a microplate reader.Optimized experimental conditions were determined by optimizing BSA concentration,aptamer concentration,and avidin-catalase concentration.The system absorbance(A550 nm)was linearly related to Salmonella typhimurcum concentration,and the detection range was from 10 to 106 CFU/mL,and the detection limit was 10 CFU/mL.