Study On Nucleic Acid Identification And Differential Diagnosis Of Brucella A19 Vaccine Strain

Background:Brucellosis is a bacterial zoonosis that seriously endangers human health.It has a history of more than 90 years since its discovery,and can be widely used in the world.In animals,it can infect mammals and wild animals,seriously harm the organs of the reproductive system,and the lesions are manifested as abortion of pregnant female animals,joint enlargement,claudication,orchitis,etc.The clinical symptoms are complex and the diagnosis is difficult.At present,the prevention and control measures are mainly vaccination of attenuated live vaccines,such as A19 and S2,which are commonly used in China.However,there are still some shortcomings,such as high toxicity of vaccines and lack of distinction between wild virus infection and natural infection.At present,the application of molecular biological detection is a popular direction.This experiment aims to establish a molecular biological differential diagnosis method for A19 vaccine strain.Objective:To establish a molecular biological differential diagnosis method for A19vaccine strain.The method can be used to detect brucellosis and distinguish brucellosis A19vaccine strain from other vaccine and wild virus strains.Mauve methods:1.Using the analysis software of A19 vaccine strains of the gene sequence analysis and 2308 wild strains,find out the differences in sequence,after using NCBI site BLAST function with other vaccine strain analysis comparison,find the difference sequence,which are very difference in sequence fragment can be brucella A19 vaccine strain testing method to establish the effective identification sequences.2.Gene sequences of Brucella A19 vaccine strain were compared with those of other vaccine strains and wild strains,and primers and probes were designed and synthesized according to the screened identification sequences.With brucella A19 nucleic acid vaccine strains,other vaccine strains and wild strains plastic recycling products as a template,establish A19 vaccine strain polymerase chain reaction(PCR)method,to optimize the conditions of the test method of annealing temperature and the specificity and sensitivity test,using the established PCR detection method with brucella Bruce method to extract DNA Ladder comparing clinical sample verification.3.Establish a multiple real-time quantitative fluorescence reaction(q PCR)detection method for A19 vaccine strain,optimize the annealing temperature,conduct specificity and sensitivity tests for the detection method,and then use the established q PCR detection method and Bruce Ladder method to compare and verify the clinical samples of DNA extraction.Results:1.The specific missing fragments of Brucella A19 vaccine strain were found through comparative analysis and screening with Mavue software,and PCR primers,double fluorescence quantitative PCR primers and probe primers were designed and synthesized according to the screened differential sequences.2.After optimizing the annealing temperature of brucella A19 vaccine strain PCR detection,the annealing temperature of A19 vaccine strain PCR detection was confirmed to be 66?.The established PCR detection method showed good specificity,only specific amplification of A19 vaccine strain was generated,and the sensitivity of PCR detection method was 3×10~2Copies each reaction.3.After the optimization of annealing temperature for the detection of brucella A19 vaccine strain q PCR,the annealing temperature for the detection of A19 vaccine strain was determined to be 68?.The established q PCR method showed good specificity,and only specific amplification for the A19 vaccine strain was generated,and the sensitivity of the q PCR method was 30Copies each reaction.Conclusion:A molecular biological detection method for Brucella A19 vaccine strain was successfully established,which is suitable for laboratory detection of brucella A19vaccine strain(PCR and real-time fluorescence quantitative PCR).Significance:The established detection method can be used to detect brucellosis affected by the interference of Brucellosis A19 vaccine strain,and effectively distinguish the A19vaccine strain from other vaccine strains and wild strains.Save time,reagent and cost,thus improve the accuracy and reliability of brucellosis detection.It is expected to play a crucial role in improving the detection efficiency of Brucellosis,so as to detect the bacteria in time,take preventive and curative measures as early as possible,stop the rapid development of the epidemic at its root,and reduce unnecessary economic losses and social security hazards.