Development Of Loop-mediated Isothermal Amplification(LAMP) Of Brucella In Regions Of Xinjiang

Object:Brucellosis is a worldwide unsolved problem,because brucellosis of livestock in manycountries and regions brought huge economic losses each year and threat to human health. Annualreport on human brucellosis more than500,000cases. Studies have shown that the ability ofpathogenic Brucella rely mainly on their ability to survive and reproduce in the host cells.Thevirulence factor which is generally found in virus is not found in Brucella. Brucella virulencefactors mainly depend on LPS, OMP and other virulence factors. OMP25is an outer membraneprotein of Brucella and omp25genes between different species of Brucella have a high degree ofhomology. Loop-mediated isothermal amplification (LAMP) has a great development in recentyears with the development of nucleic acid amplification technology.In the set of the six regions oftarget genes,we design four primers for amplification. At a constant temperature65℃in40-60min,the reaction is completed. The reaction results can be conveniently determined by the color.LAMP has convenient features and high specificity for brucellosis among cattle which can bewidely applied to the detection of great importance.Methods:First, to investigate epidemic of brucellosis in regions of Xinjiang，1721samples oflivestock were detected by agglutination test from4different territory including1053of sheepserum and668cattle serum. Brucella were isolated from stomach content of aborted fetuses at asheep farm from Wusu、Aletai、Manasi and Western Animal Husbandry in Xinjiang. Multilocusvariable-number tandem-repeat analysis(MLVA) and conventional biological identificationmethod were used in this study.Then，loop-mediated isothermal amplification（LAMP）assay wasdeveloped for rapid visual detection of Brucella. A set of six specific primers specific to regions ofomp25gene were designed. The reaction was performed in a single tube at65℃in60min withthe addition of SYBR Green I after the reaction and the result was determined by the color changeof SYBR Green I. The sensitivity and specificty of LAMP method were evaluated with theoptimized reaction system and conditions. Meanwhile, segregating strains from four regins inXinjiang were detected by LAMP and PCR.Results:The date indicated that the infection was relatively high in infected territory. Themaxlmum masculine rate was13.8%. The average masculine rate was11.1%in sheep and was4.04%in cattle.The Brucella strains was identified as B.melitensis sero type3strains by theconventional identity method and MLVA-16method displayed Brucella strain was close toBruce0261.The detection limit of the LAMP assay was about4.2fg/μL genomic DNA，about100times higher than conventional PCR method. Also,the specificity of the LAMP assay showed therewas no cross reactivity with other related bacterias.LAMP assay was evaluated by four samplesfrom brucellosis epidemic areas，and the results showed all positive samples including all PCRpositive samples. The coincidence rate of two methods was100%.Conclusions:（1）In the four regions of Xinjiang serological assay results showed that thepositive rate of brucellosis is an upward trend in these areas. MLVA-16results showed that sheepBrucella species3type,42type genotype is the predominant genotype in an endemic areas.（2）This study setted up loop-mediated isothermal amplification with gene Omp25and showed thatLAMP has high sensitivity and specificity. The coincidence rate of PCR was high.These results suggested that the LAMP assay provided a useful tool for the rapid detection ofBrucella.