A Real-time Q-PCR Method For The Detection Of Brucella And Preliminary Evaluation

Background:Brucellosis is a widespread epidemic diseases caused by Brucella infection and serious harmful to humans and mammals.In the detection and identification of Brucella,etiological,serological and biochemical methods are mainly used.However,these traditional methods have many problems such as long detection time,low positive rate,false positive and false negative results.With the development of science and technology,molecular biology technology has been increasingly used in the detection and identification of Brucella.PCR(Polymerase chain reaction)is an important method of molecular biology detection and pathogen typing of Brucella.Objective:Our study was to establish a rapid,sensitive and specific real-time q-PCR method for the detection of brucellosis,rapid clinical diagnosis,food safety quarantine and pastoral animal quarantine.Methods:In this study,a large amount of time-consuming literature search and a large number of experimental tests were to find out a Brucella DNA specific sequence,this gene sequence exists only in Brucella,but not in other bacterial viruses.We found a sequence of OMP2 in the gene pool,110 bp base length,from which the designed two primers to construct the plasmid,the establishment of real-time quantitative PCR detection method,the stability of the method for testing and sensitivity and specificity.The samples of bacilli were identified by bacterial culture and E.coli,Staphylococcus aureus,Streptococcus haemolyticus,Salmonella,Pseudomonas aeruginosa and Escherichia coli.Results:The results showed that the test has high stability between batches of tests,the sensitivity of the minimum detection limit of 20 copies/μl,the test for all species and subtypes of Brucella are amplified,and other was no cross-reaction between the non-Brucella species.Clinical trials showed that 93 cases of clinically confirmed Brucella positive specimens using the design of the PCR detection method of detection,92 cases were positive,the coincidence rate of 98.9%.In this study,the serum samples of 234 high risk patients were tested and compared with bacterial isolation and culture.The results showed that the coincidence rates were 88.46%,72.64%and 77.35%respectively.Kappa values were 0.76,0.46 and 0.54,respectively.Conclusion:The real-time PCR method was of high stability,specificity and sensitivity,and could be applied to the early diagnosis and quantitative analysis of brucellosis.