中国动物传染病学报2023，Vol.31Issue(5)：101-107,7.
改良的环介导等温扩增技术在羊布鲁菌病快速诊断中的应用
Application of Improved Loop-mediated Isothermal Amplification in Rapid Diagnosis of Brucellosis in Sheep and Goats
摘要
Abstract
Brucellosis is a serious common zoonosis that threats human health and livestock production.It is of great significance to develop establish an improved LAMP detection technique to prevent and control the spread of brucellosis.The conserved sequence of the brucellosis standard strain gene fragment was selected and primers were designed for development of a rapid LAMP detection system using on-line software.The sensitivity and specificity of the LAMP method were examined using the double ratio dilution method.Total 200 blood samples of sheep suspected with Brucella infection were tested separately by LAMP,PCR and RBPT(Rose Bengal plate test).Conformance checks were completed for evaluation of these three detection methods.The reaction system for LAMP was optimized for the final concentrations of the main substances,including MgSO4(at 8 mmol/L,Betaine at 0.8 mmol/L,calcein at 5 mmol/L and MnCl2 at 10 mmol/L.All three Brucella species were green while other 5 negative strains were orange in the specific experiment.The start time of expansion gradually extended with the dilution of the template increases.The detection limit was 10 copies/μL.The result of calcein staining was consistent with that of gel electrophoresis and PCR.Compared with PCR detection,the positive LAMP rate for 200 sheep blood samples was almost the same(P>0.05).Compared with RBPT detection,the positive LAMP rate was significantly higher than that of control group(P<0.05).In addition,there was a good agreement of PCR and LAMP test results for sheep blood samples(kappa=0.987,P>0.7).Therefore,this study provided a specific and sensitive detection kit for brucellosis.关键词
羊布鲁菌病/环介导等温扩增技术/特异性诊断/诊断试剂盒Key words
Sheep brucellosis/loop mediated isothermal amplification/specific diagnosis/diagnostic kit分类
医药卫生引用本文复制引用
于广江,张可欣,郭文平,刘金霞,郭亚春..改良的环介导等温扩增技术在羊布鲁菌病快速诊断中的应用[J].中国动物传染病学报,2023,31(5):101-107,7.基金项目
河北省教育厅重点研发项目(ZD2017101) （ZD2017101）
