为了检测绵羊脑炎李斯特菌生物膜形成情况并确定生物膜形成相关基因在该菌中的分布情况,采用结晶紫染色结合测定 OD 值的方法观察羊脑炎李斯特菌生物膜在不同时间段形成情况,并应用分子生物学方法检测生物膜形成相关基因(flaA、recA、relA、prfA、yneA、agrA、luxS、degU、motB、sigB、hpt)在该菌中的分布情况.结果表明,在37℃,绵羊脑炎李斯特菌生物膜在2 h~8 h 的 OD 值与0 h 和对照组对比差异显著(P ＜0．05),9 h~48 h OD 值呈平稳趋势,与对照组比较差异显著(P ＜0．05).2 h 开始生物膜初具形态,呈现散落的网格结构,2 h~6 h 矩阵网格结构明显,6 h~8 h 形成高度有序的网格结构,9 h~48 h依然具备生物膜网状结构特征,但个别网状结构脱落.11种生物膜相关基因在绵羊脑炎李斯特菌中的检测结果为阳性,测序结果与 GenBank 公布的相应序列同源性为87．22％~99．29％.结果表明2 h~6 h 生物膜进入黏附期和生长期,6 h~8 h 进入成熟稳定阶段,9 h~48 h 逐渐进入播散期；LM90携带 11种与生物膜形成相关的基因.%In orderto detect the biofilm formation and distribution of biofilm-associated genes of Listeria monocytogenes inducting encephalitis in sheep,the methods of crystal violet staining and OD value determi-nation were used to observe the biofilm formation in different period of time and detect distribution of bio-film-associated genes(flaA,recA,relA,prfA,yneA,agrA,luxS,degU,motB,sigB,hpt)by molecular bio-logical ways.The results showed that at 37 ℃,compared with the control group and 0 h,the biofilm OD in 2 h-8 h is significantly different (P <0.05),meanwhile,OD values in 9 h-48 h showing a stable trend is also significantly different(P <0.05).The biofilm began to be formed,showing lattice structure in the first 2 h, being more obvious for 2 h-6 h,forming highly ordered lattice structure for 6 h-8 h,maintaining the lattice structure characteristics while the individual mesh structure fell off during 9 h-48 h.1 1 biofilm-associated genes were positive in the detection,both sequence in this test and the homology published in Genbank are 87.22%-99.29%.The results showed that in 2 h-6 h the biofilm started the first step of attachment and de-velopment,in 6 h-8 h being the second one of maturation,and in 9 h-48 h being the next step of detach-ment,where LM90 carried 1 1 biofilm-associated genes.
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