Research On Identification Sequence Discovery And Differential Diagnosis Of Brucella S2 Vaccine Strain

Brucellosis is a bacterial zoonosis that seriously threatens human health.Brucella mainly includes Brucella abortus,Brucella melitensis,Brucella suis and Brucella ovis.Brucella can infect all kinds of domesticated and wild animals,and has a very serious harm to various organs of the reproductive system.It can lead to miscarriage of female animals and reduce the reproductive capacity of sick animals,causing serious economic losses to the owners?Obejective 1.Find the whole genome sequence of Brucella S2 vaccine strain,and use the genome comparison software to perform sequence comparison analysis with the wild strain 1330.Then compare and analyze with other Brucella vaccine strains to find the identification sequence of Brucella S2 vaccine strain.2.Design and synthesize primers and probes according to the identified sequence of Brucella S2 vaccine strain.Using Brucella S2 vaccine strain and the total nucleic acid of other vaccine strains and wild virus strains as templates,the conditions of PCR and multiple real-time fluorescent quantitative PCR detection methods were optimized,and specificity and sensitivity tests were established.Establish a detection method for Brucella S2 vaccine strain.Methods 1.Compare and analyze the gene sequences of Brucella S2 vaccine strain with other vaccine strains and wild strains.Primer and probe synthesis based on differential sites.2.Using the Brucella S2 vaccine strain and other vaccine strains and wild strain nucleic acids as templates,respectively,PCR and real-time PCR detection method conditions optimization,specificity and sensitivity tests were established Detection method of Brucella S2 vaccine strain.Screen the clinical samples by serological methods,extract the nucleic acid of the clinical samples and verify the established Brucella S2 vaccine strain detection method,and compare and analyze the three methods.3.The nucleic acid of clinical samples was extracted to verify theestablished Brucella S2 vaccine strain detection method,and compared with the conventional serological detection and Bruce-Ladder detection method.Result 1.Through the comparative analysis of the genome comparison analysis software,the difference sequence between the Brucella S2 vaccine strain and the wild strain 1330 was found,and then compared with the commonly used Brucella vaccine strain,it was found that Brucella can be identified.Based on the identification sequence of the S2 vaccine strain,PCR primers,fluorescent quantitative PCR primer probes and fluorescent quantitative PCR probes of non-S2 vaccine strains were designed and synthesized based on this sequence.2.After optimizing the conditions for PCR detection of Brucella S2 vaccine strains,fluorescence quantitative PCR detection and multiple fluorescence quantitative PCR detection methods,PCR detection of S2 vaccine strains,fluorescence quantitative PCR detection of S2 vaccine strains and non-S2 vaccine strains were determined.The reaction system and reaction conditions of the multiple fluorescent quantitative PCR detection of the S2 vaccine strain and the detection method have excellent specificity.The PCR detection sensitivity of the S2 vaccine strain PCR detection sensitivity is 104Copies/?L,S2 vaccine strain fluorescence quantitative PCR sensitivity is 5 Copies/?L,non-S2 vaccine strain fluorescence quantitative PCR sensitivity is 70 Copies/?L,S2 vaccine strain dual fluorescence quantitative PCR sensitivity is 200 Copies/ ?L.Conclusion Successfully found the identification sequence that can distinguish the Brucella S2 vaccine strain from other vaccine strains and wild strains,and established the detection method of the Brucella S2 vaccine strain based on this sequence,including conventional PCR detection and fluorescent quantitative PCR detection And multiple fluorescence quantitative PCR detection.The detection method can effectively detect whether the brucella S2 vaccine strain is infected and is used for the detection of brucellosis interfered by the Brucella S2 vaccine strain.Thereby improving the accuracy and reliability of brucellosis detection.