Establishment Of Method And Application Of VirB8-PCR For Detecting Of Brucella And Sequence Analysis On Erythritol Catabolic Genes

To develop a method detected the virulence factor VirB8 of brucella for useing in diagnosis and surveillance of brucellosis. The virB8 gene from type IV secretion system(TTSS)of brucella was used as template and the primers were designed according to the sequence of virB8 gene in GenBank. The reaction condition and program for the VirB8-PCR were optimized, and this method of assay was used for detection of the virB8 gene in standard reference stains of Brucella and in the isolates on local area. It was found that the specificity of this assay was 90% and the sensitivity was 153 fg DNA(corresponding to 30 bacteria)respectively. And this assay was good at reproducibility and stability, the keeping time of premix fluid at -20℃is over 6 month. VirB8 genes were amplified from 7 different strain of brucella by PCR, and The PCR products were cloned in pMD18-T vector for sequencing. Sequence were analysis to show that the VirB8 genes of 7 different brucella strains compared with standard sequence the nucleotide homology above 99.3%, which revealed that the specificity of the VirB8-PCR is very reliable.In order to investigate the occurrence and epidemic of brucellosis, a total of 1354 domestic animal(713 cattles and 641 sheeps)to come from 4 farms, tested by RBT, SAT, iELISA, cELISA and PCR for survey. The results showed that cattles were positive for brucella was 30.2%, sheep was 19.7%, compared with the past there was an obvious increasing trend. Based on the survey, samples were collected and used for bacterium isolation. There were 49 strains of doubtful bacteria were isolated by using a selective culture medium. 13 strains were identified as brucella by VirB8-PCR, 12 strains were B.abortu, and 1 strains B.melitensis, which was B.melitensis III identified by AMOS-PCR and a serial biochemical tests.The primers were designed for amplification of brucella erythritol catabolic genes (Ery) according to the complete genomic sequence of B. abortus 2308, published in GenBank. Ery genes were amplified from 3 strains different B.abortus 19 by PCR. The PCR products were cloned in pMD18-T vector for sequencing. Sequence analysis showed that the Ery of reference strains was absent on 702bp, and 2 of Chinese strains had no basic pair absented, compared for 2308 strain, but in the sequence:at 51st, 52nd, CG changed to GC;at 521st, 522nd, 523rd, losted AGG;at 547th,T becomed C. Caused 3 amino acid changed:at 13th,R become A;at 170th, T become A;at 178th, V become A.