Establishment Of Fluorescence PCR For Detection Of Brucella

Brucellosis is one of the most serious zoonoses in the world.The traditional detection methods for Brucellosis have low positive rate and high operational risk,while the current serological detection techniques have certain limitations in sensitivity and specificity.In addition,due to the limited types of serological detection samples,it is impossible to monitor brucellosis in the environment.Therefore,the development of a rapid,sensitive and accurate detection method for brucellosis and type monitoring for human and animal animals and their living environment,animal products and other potential sources of infection can provide strong support for the prevention and control of brucellosis.Methods:Based on the highly sensitive and specific q PCR detection technology,the primer probe was designed for the single copy gene Bcsp31 gene of Brucella,and the relatively conservative sequence of mammalian in mitochondrial 16S gene was used as the internal standard to design the primer probe.The partial sequences of ALk B/IS711 and BMEI1162/IS711 of Brucella abortus and Brucella melitensis were used to design primers and probes to establish a q PCR method for the identification of Brucella abortus and Brucella melitensis.The two reaction systems and conditions were optimized,and the sensitivity,specificity and repeatability were verified,and the standard curve was made.Finally,the two reaction systems were verified by actual samples.Result:1.Established and optimized the detection method of Brucella by fluorescence PCR.The standard curve was y=-3.459（lgx）+46.962,and the correlation coefficient R~2was 0.999.The detection limit can be as low as 550copies/m L.The detection limit can be as low as 550copies/m L.There was no cross reaction with Mycobacterium tuberculosis,Salmonella typhi,Escherichia coli and Yersinia,and the reaction system had good specificity.Within the linear range,the repeatability between batches and within batches were less than 2%,and the reaction system had good reproducibility.2.Mammalian mitochondrial 16S gene was used as internal standard for quality control of PCR reactionIn addition to the human samples,it was significantly amplified in samples of cattle,sheep,pigs,dogs and mice.In addition,the nucleic acids extracted from whole blood,serum,feces,urine,saliva,vaginal secretions,semen and milk samples also had obvious amplification reaction.Therefore,the internal standard can effectively monitor the nucleic acid extraction and PCR reaction of human and animal Brucella samples,and avoid false negative results caused by reaction inhibitors and not extracting nucleic acid.3.Established and optimized the identification method of Brucella abortus and Brucella melitensis by fluorescence PCR.The standard curve of Brucella in cattle was y=-3.301（lgx）+45.602,and the correlation coefficient R~2was 0.998;the standard curve of Brucella melitensis was y=-3.157（lgx）+46.583,and the correlation coefficient R~2was 0.99.The detection limit of Brucella abortus and Brucella melitensis reached600copies/m L and 700copies/m L,respectively,and the linear range was 10~9-10~4copies/m L,and the repeatability CV values of each concentration in the linear range were less than 2.1%,with good reproducibility.There was no cross reaction between the reaction system and the Brucella negative reference bacteria.Among them,the VIC chanel have specific amplification to Brucella melitensis,but have no specific reaction to Brucella abortus and Brucella sus;the FAM chanel have specific amplification to Brucella abortus,but have no specific reaction to Brucella melitensis and Brucellasus.The reaction system has good specificity.4.The reaction system was applied to the actual sample detection,a total of 263blood samples of suspected brucellosis patients were detected,and the Brucella detection results were compared with RBPT＆SAT（the diagnostic standard of brucellosis）.5.In the samples,63 cases of Brucella melitensis and 1 case of Brucella abortus were detected by fluorescent PCR.The positive samples were mainly Brucella melitensis infection.The results were basically consistent with AMOS-PCR method.Conclusion:This experiment established a sample for human directly detect brucella fluorescence PCR detection method,for the subsequent development of brucella fluorescence PCR detection kits established the foundation,in addition to Brucella melitensis and Brucella abortus in identification,this method is rapid,sensitive,specific,and relatively safe,has a good application prospect.