LAMP技术快速诊断布鲁氏菌病的研究  

Rapid Diagnosis of Brucella by Loop-mediated Isothermal Amplification

作　　者：谢文萍[1] 肇慧君[2] 张琳[2] 姜红旭[2] 吴斌[2] 孙浩[1] XIE Wen-ping ZHAO Hui-jun ZHANG Lin JIANG Hong-xu WU Bin SUN Hao(Dalian Polyteehnic University, Dalian 116034 Liaoning Entry-Exit Inspection and Quarantine Bureau, Dalian 116001)

机构地区：[1]大连工业大学,大连1160341 [2]辽宁出入境检验检疫局,大连116001

出　　处：《生物技术通报》2017年第3期186-192,共7页Biotechnology Bulletin

基　　金：国家质量监督检验检疫总局科研项目(2014IK248)

摘　　要：旨在应用环介导等温扩增技术(Loop-mediated isothermal amplification,LAMP)对布鲁氏菌进行研究。针对布鲁氏菌保守基因16S r DNA设计LAMP引物,通过浊度法对LAMP反应条件进行优化,应用建立好的LAMP反应条件进行特异性及灵敏性试验。结果显示:(1)优化后的反应条件是62℃恒温60 min,内引物1.50μmol/μL、外引物0.20μmol/μL、环引物1.0μmol/μL。(2)该LAMP方法与3株布鲁氏菌均发生了扩增反应,达到阳性浊度值,而与小肠耶尔森(ATCC9510)、大肠杆菌(ATCC25922)、沙门氏菌(ATCC10708)和金黄色葡萄球菌(ACTT33591)等标准菌株没有扩增反应。(3)浊度法和实时荧光法两种方法的检测最低限分别为4.36 fg/μL和436 fg/μL,其灵敏度均高于普通PCR的最低检出值4.36 pg/μL。LAMP检测技术用于布病临床诊断具有快速、高效、便捷等特点,对于基层布病的病原学诊断具有实用价值。This study aims to apply the loop-mediated isothermal amplification （ LAMP ） in detecting Brucella. A set of six specific primers specific to regions of 16S rDNA gene were designed, and the turbidity technique was employed to optimize the reaction conditions, by which the specificity and sensitivity of the method were evaluated. Results are ： （ 1 ） the optimized temperature was 62% at constant 60 min, and the concentration of inner primer was 1.50 μmol/μL, 0.20 μmol/μL outer primers, and 1.0 μmol/μL loop primers ; （ 2 ） furthermore, 3 strains of Brucella occurred LAMP amplification reaction and achieved the positive turbidity value, but there was no amplification with other control group including Yersinia enteroeoliticu ATCC9510, Escherichia coil ATCC25922, Salmonella typhimurium ATCCI0708, and Staphylococcus aurens ACTT33591 ; （ 3 ） the minimum thresholds for turbidity technique and real-time fluorescence were 4.36 fg/μL and 436 fg/μL respectively, their sensitivities were both higher than the value by conventional PCR, 4.36 pg/μL. Obviously, it is fast, efficient, and convenient while LAMP is applied in the clinic diagnosis of Brucella, therefore, it is practically applicable for the pathogenic diagnosis of Brueella in grassroots communities.

关 键 词：布鲁氏菌 16S RDNA 环介导等温扩增技术(LAMP) 

分 类 号：R516.7[医药卫生—内科学] R440[医药卫生—临床医学]