采用环介导等温扩增技术快速检测猪丹毒丝菌病原体

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 Isotherml 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200CFU(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 1 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. rkwiopathiae , 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 CFTJ/ mL). The detection limit of LAMP and PCR for the E. rhusiopathiae in infected mice blood were 200 CFU (10~7)/raL. [ Conclusion ] LAMP technique combined with FTA Filter is a potential method for rapid detection of E. rhusiopathiae.