Study On The Reference Materials Of Preparation About Salmonella Enteritidis In Chicken And Nucleic Acid

Objective:Salmonella enteritidis (SE) is a Common pathogenic bacteria that cause of food poisoning,and it has great significance in the Public Health.Poultry, eggs and meat products are the main media of the SE, it make a serious impact on the development of aquaculture and human health.Therefore those food that contaminate SE vulnerable before into the market should make rigorous testing. To ensure the inspectors’capability, standardize the management of the kit, and to reduce the leak detection and error detection rate in food,we should increase efforts to develop the Biological Characteristics Reference Materials,so that ensure the results true and reliable.The test launched a series of studies of preparation of SE reference materials in chicken and SE nucleic acid reference materials.On the basis to add matrix in Biological reference materials to make the test conformity to the actual value more.Supply a reference value for the same type of reference materials’preparation in future.Method:The test is divided into two test sections, Test1:Preparation of SE reference material. Test2Preparation of SE nucleic acid reference material.Test l:Detection of antibiotic residues in the pre-added chicken matrix and detection the matrix impact on SE;Take sterilization,the cooked chicken matrix take high-pressure steam sterilization and the raw chicken matrix takeé'´-60sterilization,to achieve the requirements of sterile matrix;Develop the dehydration test about the two chicken matrix,to determine the amount of water used for recovery;To draw the growth curve,as a reference for the broth culture time,and use the broth witch in the mid-term of growth to preparation of reference material;Study on screening the Lyoprotectant for reference material and choose the best pre-freezing method to assure the highest survival rate after freeze-dried;Test the uniformity stability, tracking, and assessment of uncertainty of reference material.SE-RM in chicken were conducted six batches of preparation, each batch prepared the SE-RM containing100bottles of cooked chicken matrix and100bottles of raw chicken matrix.The results of the uniformity experiment showed that, the repeatability standard deviation of raw chicken matrix and cooked chicken matrix is2.185%and3.596%,the reproducibility standard deviation is2.185%and3.514%,the results of the Analysis of Variance are P>0.05,it indicated that there was no significant difference between the six batches of RM. Stability experiments showed that the RM stord at-20℃,the microbial content of raw matrix remain800CFU/g,the stability test of cooked matrix appear a small amplitude fluctuations,its stability should to continue to track.The uncertainty of electronic balance is2.7934mg,so it can be neglected.And the uncertain of the constant value methods about raw chincken matrix and cooked chicken matrix is11and8,and the two reference material with difference matrix’s final value is:value±11,value±8.Test2:Choose the method to extraction SE genome (gDNA),test the concentration, purity,characterized and sequencing.Than diluted gDNA to10g/mL,dispensed into vials,100μL/bottle.After freeze-drying process,store at-20℃.Reference material uniformity test and tracking the stability of reference material.Evaluation of uncertainty of the gDNA reference material.In total390products of the gDNA-RM were developed final. Each bottle containing the gDNA about1μg. Test its uniformity and track its stability,and establish to the assessment of the uncertainty.Use The Picofluor TM portable fluorescence to measured the content of gDNA in sample,and the uniformity analysis showed that Fvalue=0.8357<F0.05(14,30)=1.68,so the content of gDNA is uniform. In the Stability experiments,the samples were grouped and stord at-20℃,4℃,25℃and60℃respectively,track the content of gDNA in RM,genomic integrity and characteristics of gDNA.The short-term stability hold7weeks and the long-term stability hold6months,the results show that the RM stored at-20℃,4℃,25℃temperature, short-term and long-term preservation has good stability,and stored at60℃, the RM is very unstable, but the invA gene can also was amplified.Conclusion:The Study establish a Preparation method of SE reference material and SE nucleic acid reference material.The uniformity are good, and store at-20℃, the reference material have a good short-term stability. The long-term stability of the reference material generally need to be tracked for2years, but due to time constraints, until the papers submitted it can not complete the long-term stability test, stability needs to continue track in order to ensure the effectiveness of the reference material’s development. In addition, because the study aim to investigate the process about preparation of reference material,therefore the amount of preparation can meet its objectives directiy.