Objective To establish a real-time fluorescent quantitative polymerase chain reaction assay system for rapid and accurate detection of Brucella and to provide a guidance for clinical doctors in making diagnosis of Brucella infection.Methods A real-time polymerase chain reaction assay was developed for detection of all species and bivors of Brucella.According to the sequences of Brucella surfaceprotein gene 31,a probe and two primers were designed.The specificity (Brucella standard strains 16M,544A,1330S,vaccine strains 104M,reference strains Yersinia enterocolitica O:9,E.coli O:157) and repeatability (4 different concentrations of standard) were verified and the lowest limit of detection of the reaction system was analyzed.Results The established method can be used for specific amplification of Brucella,the different substrate concentrations of the reaction system showed good linearity with the Ct value (Y =-2.77X + 43.19,R2 =0.994 8),the lowest limit of detection of reaction system was 1 × 103 copies/ml,the test results of Brucella standard strains 16M,544A,1330S and 104M were positive,test results of Yersinia enterocolitica O:9 and E.coli O:157 were negative,4 different concentrations of standard and negative qu ality control were tested 4 times,and the variation coefficients were 0.15％,0.16％,0.23％ and 0.18％,respectively.Conclusion A Brucella real-time fluorescent quantitative polymerase chain reaction assay system is established successfully,which could be used to identify Brucella rapidly and accurately.

Objective To establish an accurate testing method for detection of Brucella and Brucella Abortus,Brucella Melitensis,Brucella suis.Methods The strains include the standard Brucella melitensis 16M,Brucella abortus 544A,the standard Brucella suis 1330s,and the clinical specimens Brucella melitensis 81190,Brucella abortus 80176,104M,Brucella suis 80177,19971,19972.All 9 strains were saved at the Base for Prevention and Control of Plague and Brucellosis,the Chinese Center for Disease Prevention and Control.DNA was extracted and used to amplify by PCR,Yersinia pestis O ∶ 9 served as control.The single nucleotide polymorphisms probes were designed on the basis of twosites (379,576) in conservative segment from Blucella flhA,and made into gene chip.According to part sequence of flhA,primers were labeled by biotin,target gene was amplified by PCR,and hybridized with the designed gene chip,and finally,biotin was used for identification.Results The length of all 9 strains were all 357 bp,consistent with expected results.The Yersinia pestis O ∶ 9 that had the same antigens with the Brucella showed no specific PCR products.At the 379 and 576 points of gene,the Brucella suis was 379G,576C,the Brucella Abortus was 379T,576T,the Brucella Melitensis was 379T,576C,all these results were identical to the designed probe sequence.Experimental verification of membrane gene chip,the Brucella suis showed color at the points of 379G,576C and F,the Brucella Abortus showed color at the points of 379T,576T and F,the Brucella Melitensis showed color at the points of 379T,576C and F.Conclusion Brucella gene chip can be used to identify Brucella rapidly.