Establishment Of Real-time Fluorescence Quantitative PCR For Rapid Detection Of Brucella And Its Preliminary Application

Objective To establish a real-time fluorescence quantitative PCR technique for rapid screening and assistant diagnosis of Brucella,and to apply it for early detection and surveillance of Brucellosis.Methods: 1.According to the base sequence of Brucella outer membrane proteins omp10 and omp31,four pairs of primers and probes that were omp10,omp10-1,omp31 and omp31-1,were designed by Primer Premier 5.0 and synthesized by Shanghai Huirui Biotechnology Limited Company.2.The primers and probes were used to detect 19 strains of 6 categories Brucella DNA of known organisms,10 strains of Brucella DNA that were isolated from Yuxi of Yunnan.And 224 negative Brucella DNA,including 35 strains of Bartonella DNA,103 strains of the Lord Komori enterocolitis DNA(including 4 parts of O: 3 and 9parts of O: 9)and 86 samples of hybrids bacteria DNA.Then evaluating the specificity of primers and probes based on the test results.3.The established rapid detection technique was used to detect the DNA of 20 strains of Brucella isolated from Yunnan,20 whole blood DNA of Brucella antibody positive patients and 160 whole blood DNA of suspected Brucella patients.Results 1.The DNA of 19 standard Brucella strains could be amplified by omp10 and omp10-1,and the Ct value of omp10 was less than omp10-1.Omp31-1 could amplify the DNA of 16 standard Brucella strains.The DNA of standard Brucella strains could not be amplified by omp31.2.The DNA of 10 strains of Brucella which were isolated from Yuxi of Yunnan could be amplified by omp10,omp10-1 and omp31-1,and the DNA of negative control strains could not be amplified by omp10,omp10-1 and omp31-1.3.The rapid detection technique based on OMP10 primer and probe can amplify the DNA of 20 strains of Brucella isolated from Yunnan and 20 whole blood DNA of brucellosis patients,and partly amplify the DNA of 160 suspected brucellosis patients.Conclusion The real-time fluorescence quantitative PCR detection technology based on omp10 primer and probe can be used for rapid detection of Brucella and surveillance of brucellosis epidemic,as well as clinical auxiliary diagnosis.