| With the extensive use of antibiotics,the drug resistance of Salmonella is becoming more and more serious,which poses a great threat to human and animal health.For a long time,a large number of scholars and government departments have done extensive investigation and monitoring on the drug sensitivity of food borne Salmonella and the coding genes carried by drug-resistant strains,but there is still a lack of standard samples used as reference in drug resistance gene detection.In order to better,faster and more accurately understand the resistance genes and related resistance mechanisms of foodborne pathogens,it is necessary to use standard strains in the detection process.In this study,Salmonella strains with high drug resistance and stable resistance genes will be screened,and then reference strains suitable for detection of food borne pathogens carrying β-lactamase coding genes will be prepared.The development of standard samples of such strains will realize the wide application of standard samples of bacteria strains with independent intellectual property rights in the field of food drug-resistant microorganism standard samples,and solve the problems related to national standards The problem that reference strains are occupied by foreign strains for a long time in the detection can save the foreign exchange expenditure of standard samples of drug-resistant microorganisms in food.In this study,Gram staining,16S rDNA sequencing,Vitek automatic biochemical detection system and MALDI-TOF-MS protein identification were used to identify the candidate strains,which were put on record in the relevant national institutions and applied for the development of national standard materials;vacuum freeze-drying technology was used to prepare reference strain samples;the prepared samples were homogenized and stored at 37℃ 13 days,4℃ 90 days and-80℃ 360 days were used to monitor the storage stability of samples,the genetic stability of drug resistance genes and phenotypes.At the same time,six laboratories which meet the requirements of testing ability,such as testing personnel,reagent equipment,environment and traceability,were selected for joint calibration.The main achievements are as follows1.Identification of strains and application for record in relevant national institutions:118 strains of Salmonella with different drug resistance phenotypes and levels were selected from the strains isolated in the early stage of the laboratory;Gram staining,16S rDNA sequencing,Vitek and matrix assisted laser desorption ionization time of flight mass spectrometry were used The 118 strains were identified by morphology,molecular biology,physiological and biochemical reactions and protein mass spectrometry.Three strains carrying β-lactam resistance genes were screened,namely,blactx-m-55 and blatem-11 strains carrying resistance genes and blactx-m-3 strains carrying resistance genes alone There were 1 strain carrying drug resistance genes blatem-1,blaoxa-1 and blactx-m-151,5 strains with high drug resistance phenotype,including ampicillin(AMP)512 μg/ml,streptomycin(STR)128 μ g/ml,chloramphenicol(CHL)128 μ g/ml,cefoxitin(Fox)64 μg/ml and gentamycin(Gen)32 μ g/ml 1 strain;obtained the CMCC number of China Medical bacteria preservation center;submitted to the National Standards Committee for the development of reference strains.2.Preparation of reference strains by vacuum freeze-drying technology:8 reference strains were prepared in the Chinese Academy of inspection and quarantine,and vacuum freeze-drying technology was used for the vacuum packaging of penicillin bottles.3.The genetic stability,homogeneity and storage stability of 8 candidate reference strains were monitored.The results showed that the number of viable bacteria was about 104 CFU/bottle after stored at 37℃ for 13 days,106 CFU/bottle after stored at 4℃ for 90 days and 3 × 106 after stored at-80℃.for 360 days CFU/bottle,and the drug resistance genes and phenotypes can be inherited stably in the preservation process,with good uniformity,which can be used as positive strains for drug resistance detection.4.The feedback result of the combined set value is good,which meets the requirements of the preparation of reference strains. |
