Study On Multiplex PCR Detection Kits For Salmonella And Staphylococcus Aureus In Agricultural Products Processing

Salmonella and staphylococcus aureus are the check project of our country agricultural product quality safety control.In the process of agricultural products processing,pathogenic bacteria are influenced by all kinds of processing technology(temperature,chemical,etc),kill pathogenic bacteria in the state,easy to produce false-negative results,and cause the leak detection.In view of the agricultural products processing in salmonella and staphylococcus aureus detection requirements in China,texting the two kinds of target pathogens in the different complex substrate of agricultural products,using molecular biology techniques,setting up two kinds of rapid detection method of bacterial pathogens in the same system,to meet the needs of the actual testing.This study aimed at every link of the whole testing process optimization speed,in order to achieve fast and efficient detection purposes.Related experiment is as follows:1.The development of enrichment-repair medium of salmonella and staphylococcus aureusResearch the enrichment-repair medium of salmonella and staphylococcus aureus in the agricultural products processing,and using common enrichment-repair medium to detect produce with artificial inoculating two kinds of target bacteria,to observe the influence of different agricultural products matrix training target bacteria in culture media.Determine the formula of the common enrichment-repair medium:tryptone 10 g,yeast powder 5g,soya peptone 25 g,NaCl 10 g,glucose 4g,salicylic acid 2g,sodium pyruvate 2g,ox-gall salt 0.3g,potassium tellrite 0.3mg,LiCl 0.5g,Twain-80 1mL,distilled water 1000 mL,pH7.2±0.2.Set up the damage model of salmonella and staphylococcus aureus,by using the methods of heading,refrigeration and acid damage to make salmonella and staphylococcus aureus in sub-lethal injury.When the target bacteria in frozen-80 for 30 d,℃ the sub-lethal injury rate is highest,Salmonanella is 19.35% and staphylococcus aureus is 17.26%;the effect of common enrichmentrepair medium to repair the injury target pathogens is good,the repair time of thermal damaged target bacteria is 2h,the repair time of cold stress and acid damaged target bacteria is 4h.Verify the enrichment effect of the common enrichment-repair medium.After training 4h,bacteria liquid concentration: salmonella is 2.2×103CFU/mL,staphylococcus aureus is 3.7×103CFU/mL,achieving the limit of rapid detection.When salmonella and staphylococcus aureus in a ratio of 1:1(10CFU/mL)co-culture for 4h,salmonella’s bacteria liquid concentration is 6.25×104 CFU/mL and staphylococcus aureus is 4.23×104CFU/mL.When salmonella and staphylococcus aureus in a ratio of 1:10(10±1CFU/mL:100±10CFU/mL)co-culture for 4h,salmonella’s bacteria liquid concentration is 2.1×104 CFU/mL and staphylococcus aureus is 1.9×106CFU/mL.When salmonella and staphylococcus aureus in a ratio of 10:1(100±10CFU/ mL:10±1CFU/mL)co-culture for 4h,salmonella’s bacteria liquid concentration is 1.02×106 CFU/mL and staphylococcus aureus is 1.4×104CFU/mL.When the ratio is 1:100(10±1CFU/mL: 1000±100CFU/mL)co-culture for 6h,salmonella’s bacteria liquid concentration is 2.45×103CFU/mL and staphylococcus aureus is 1.5×103CFU/mL.Through the validation for the enrichment effect of the common enrichment-repair medium,whether it is a single bacterium enrichment cultivation or composite enrichment,to satisfy multiple PCR detection limit with cultivating 4-6h.At the same time,common enrichment-repair medium inhibit the growth of the non-target bacteria.Pollut food samples,common enrichment-repair train directly only after filtering processing of food samples.After training,the discovery is that compare with the pure microbial cultivating 4h,after 6h food samples of artificial pollution target bacteria were cultured,bacteria liquid concentration can reach 104-105CFU/mL,achieve multiple PCR detection limit.Don’t need to make special treatment for matrix of agricultural products,can directly detect.2.Research the genome DNA extraction method of salmonella and staphylococcus aureusTo extract Salmonella and Staphylococcus aureus DNA by the methods of Boiling-WaterExtraction,kit assay,improved CTAB method,DNA extracting solution method and TEX cracking liquid method.The results showed that TEX cracking liquid method to extract high purity of DNA,simple operation,save time and effort,low costs.The purity of Salmonella DNA is 1.88,and the concentration is 412.6ng/μL;the purity of Staphylococcus aureus DNA is 1.84 with the concentration 366.7ng/μL by using the TEX cracking liquid method.The extraction of DNA by PCR can amplify objective band with extraction DNA as template.3.Set up multiple PCR detection system for Salmonella and Staphylococcus aureusIn the system,choosing salmonella invA as target gene,the primer sequence is :forward primer 5’-GTCATGATATTCCGCCCCATATT-3’,reverse primer 5’-CGGTGCGATGAAGTTT ATCAAAG-3’;choosing staphylococcus aureus nuc as target gene,the primer sequence is :forward primer 5’-GCTCAGCAAATGCATCACAAAC-3’,reverse primer 5’-AGCGTTGTC TTCGCTCCAAA-3’.After optimizating annealing temperature and concentration of primers,the annealing temperature is 56 and the concentration of primers is ℃ 0.1μmol/L.Multiple PCR detection sensitivity: salmonella 1.7×103CFU/mL,staphylococcus aureus 1.3×103CFU/mL.4.The development of Salmonella and Staphylococcus aureus in agricultural products processing by multiple PCR detection kitsAfter the optimization and assembly of reaction system,developing multiple PCR detection kits for salmonella and staphylococcus aureus in agricultural products,to evaluate the performance of the kit.The specificity experiment of the kit results show that the kit can accurately detect salmonella and staphylococcus aureus and no cross reaction with other non-target bacteria.The sensitivity experiment of the kit results show that the limit of the kit is salmonella 1.7×103CFU/mL and staphylococcus aureus 1.3×103CFU/mL.The repeatability experiment of the kit results show that the repeatability is good between kit batches and kits batch.The stability experiment of the kit results show that it is no significant influence to the kits after repeated freezing and thawing process.Kits can avoid light preservation nine months at-20℃.To test the actual samples using the kit,the detection rate of salmonella and staphylococcus aureus was 6.7%(14/210)and 9.5%(20/210)in 210 samples.The test results are in conformity with the test results of national standard method,coincideence rate was 100%.In practices,kit method can get the results about 4-12 h,the national standard method takes more than 5-7d to get the conclusion.The kits have the advantages of strong specificity,high sensitivity,good repeatability and strong atability,they are suitable to detect rapidly salmonella and staphylococcus aureus in the processing of agricultural products.