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 Amplifi cation in Rapid Diagnosis of Brucellosis in Sheep and Goats

作　　者：于广江 张可欣 郭文平 刘金霞[1] 郭亚春[1] YU Guangjiang;ZHANG Kexin;GUOWenping;LIU Jinxia;GUO Yachun(Experimental Center of Pathogen Biology,Chengde Midical University,Chengde 067000,China)

机构地区：[1]承德医学院病原生物学实验中心,承德067000

出　　处：《中国动物传染病学报》2023年第5期101-107,共7页Chinese Journal of Animal Infectious Diseases

基　　金：河北省教育厅重点研发项目(ZD2017101)。

摘　　要：布鲁菌病严重威胁人类健康和畜牧业生产的常见人畜共患病。建立改良的LAMP,对防控布鲁菌病的传播具有重要意义。选择羊布鲁菌标准菌株DNA保守序列,设计LAMP引物;建立并优化LAMP快速检测体系;通过倍比稀释法等完成灵敏度和特异性实验;选择可疑布鲁菌感染羊血液样本200例,分别进行LAMP、PCR和RBPT检测,完成一致性检验。LAMP反应体系中MgSO_(4)为8 mmol/L,甜菜碱为0.8 mmol/L,钙黄绿素为5 mmol/L,MnCl_(2)为10 mmol/L;布鲁菌均呈现绿色,5种阴性菌株均为橙色;随着模板稀释度的逐渐增大,扩增开始时间逐渐延长,检测极限为10 copies/μL,与凝胶电泳、PCR结果一致;与PCR比较,羊血液标本LAMP阳性率基本一致,无统计学意义(P>0.05);与RBPT比较,LAMP阳性率显著升高,有统计学意义(P<0.05);PCR和LAMP检测一致性较好(Kappa=0.987,P>0.7)。因此,本研究为布鲁菌病提供了特异性好,灵敏度高检测试剂盒。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 withBrucellainfection 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 thefinal concentrations of the main substances,including MgSO_(4)(at 8 mmol/L,Betaine at 0.8 mmol/L,calcein at 5 mmol/L and MnCl_(2) at 10 mmol/L).All threeBrucellaspecies 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 

分 类 号：R378.91[医药卫生—病原生物学]