采用环介导等温扩增技术快速检测猪丹毒丝菌病原体  

Rapid Detection of Erysipelothrix rhusiopathiae Using Loop-Mediated Isothermal Amplification

作　　者：克力比努尔.热合曼 杨春静[1] 晏鹏飞[1] 李江伟[1] 

机构地区：[1]新疆大学生命科学与技术学院/新疆生物资源基因工程重点实验室,乌鲁木齐830046

出　　处：《新疆农业科学》2012年第7期1321-1327,共7页Xinjiang Agricultural Sciences

基　　金：新疆生物资源基因工程重点实验室开放基金(XJDX0201-2011-04)

摘　　要：[目的]建立猪丹毒丝菌(E.rhusiopathiae)毒力株的快速检测方法。[方法]利用快速灵敏的环介导等温扩增技术(Loop-mediated Isothermal amplification,LAMP)针对丹毒丝菌spa基因较为保守区域设计4条特异性LAMP引物,采用FTA(Flinders technology associates,FTA)滤膜法提取12种血清型丹毒丝菌,8种非丹毒丝菌及受感染小鼠血液的基因组DNA,分析LAMP环引物的特异性;比较LAMP与传统PCR方法的扩增敏感性和产物特异性。[结果]在61℃恒温条件下,LAMP法能够特异性扩增丹毒丝菌特异条带,与常规PCR扩增结果一致;灵敏度实验表明LAMP法检测猪丹毒丝菌的检测下限为2 CFU/mL,较传统PCR的敏感性(大于20 CFU/mL)至少高10倍。LAMP方法与PCR方法都能在小鼠感染模型血样中检测到浓度为200 CFU(10^(-7))/mL以上浓度的丹毒丝菌。[结论]LAMP法与FTA滤膜法相结合在检测丹毒丝菌中具有潜在的应用价值。[Objective]To develop rapid and convenient method for detecting E.rhusiopathiae. [Method]LAMP was applied to amplify the gene fragment from the genomic DNA of E.rhusiopathiae extracted with FTA filter by using the four specific primers targeting the conserved region of spaA gene.Twelve Erysipelothrix spp strains and eight non - Erysipelothrix spp strains were detected with LAMP and PCR method to compare the sensitivity and specificity.The E.rhusiopathiae extracted from the infected mice blood was detected by the LAMP and PCR as well.[Result]Results showed that the target gene was amplified specifically with loop - primer of LAMP at 61℃isothermal condition.The sensitive of LAMP for detecting the E.rhusiopathiae was 2 CFU/mL which was 10 times higher than that detection with PCR(more than 20 CFU/ mL).The detection limit of LAMP and PCR for the E.rhusiopathiae in infected mice blood were 200 CFU (10^(-7))/mL.[Conclusion]LAMP technique combined with FTA Filter is a potential method for rapid detection of E.rhusiopathiae.

关 键 词：丹毒丝菌 spaA LAMP FTA 

分 类 号：S852.6[农业科学—基础兽医学]