Establishment Of Real-time Multiplex Fluorescence PCR For Rapid Identification Of Brucella

Aplication real-time multiple PCR technology to establishment rapid identification method of brucellosis。According to the specific gene BCSP31 of brucella,ALKB/IS711 and BMEI1162/IS711 target genes as part of the fragments,primers and probes are designed and establish multiple real-time PCR amplification syetem。real-time multiplex PCR detection of Brucella showed that only brucella.,spp B.abotus and B.melitensis have fluorescence signal,the other biological-type Brucella strains and with a higher homology pathogens were no fluorescent signal。The amplified product as target gene are cloned to PUCm18-T vector,preparation of standard sample and standard curve。serial dilution of sample were Amplificated to determine the sensitivity of this method。Application other biological-type strains of Brucella and homology closer to pathogens (Hansebaer quintana,Vibrio cholerae,Francisella tularensis,Escherichia coli O:157,E. coli O:16,small intestine colon Yersinia O:9,Salmonella group N,serotypes of Pseudomonas maltophilia) to verify specificity of test。DNA were extracted from 97 Brucella strains,So as to achieve the purpose of rapid identification of Brucella。The test of single,multi-detection sensitivity were 7.68,7.58,7.54×102copy/μl or 13~24fg/μl;while the this primers conventional PCR detection sensitivity 7.68,7.58,7.54×104copy/μl or 130~240pg/μl。the method can specific identify the Brucella,B.abotus and B. melitensis and has higher sensitivity,quick and Convenient operation.,et al;can be used for the rapid identification of Brucella,disease monitoring and pathogen outbreak investigation。...