单核细胞增生李斯特菌检测技术研究进展

单核细胞增生李斯特菌(Listeria monocytogenes,LM)是常见的食源性致病菌之一。目前,在众多单增李斯特菌的检测方法中应用较广的是免疫学检测法、分子学检测法。免疫学检测时间短,操作简单,但该方法依赖高特异性的抗体,会出现假阳性,还需要进一步鉴定检测结果。分子学检测法克服了免疫学检测法不能在种的水平鉴定单增李斯特菌的缺点,省时省力,灵敏度高,但是分子学检测法需要丰富的操作经验,并且不适于现场大批量检测。新兴的代谢学检测法、光谱学检测法、生物传感器等也都有各自的优缺点。本文综合近年最新文献,就单增李斯特菌检测的最新方法、检测进展及未来发展趋势予以分析综述,以期为该菌的检测提供参考。     

单核细胞增生性李斯特菌及其快速检测

李斯特氏菌是一类人畜共患病的病原体 ,其中以单核细胞增生性李斯特氏菌 (Ｌｉｓｔｅｒｉａｍｏｎｏｃｙｔｏ ｇｅｎｅｓ ,ＬＭＯ)为人们所重视 ,这种菌可引起多种畜禽和人类疾病 ,本文从ＬＭＯ的生物学特性、流行病学角度出发 ,来介绍ＬＭＯ的传播特性及其预防、快速检测方法。1　ＬＭＯ生物学特性和流行病学特性ＬＭＯ是革兰氏阳性短小杆菌 ,需氧或微嗜氧 ,2 0～ 2 5℃时有动力 ,对温度适应性较强 ,1～ 4 5℃内均可繁殖 ,较低温度下比其他细菌长得更好 ,并且毒性更大 ,该菌耐热 ,经过巴氏消毒后 ,部分细菌仍可存活 ,对亚硝酸盐、食盐及低 …     

棉铃虫作为单核细胞增生李斯特菌感染模型的初步研究

研究单核细胞增生李斯特菌(Listeria monocytogenes,简称Lm)对棉铃虫(Helicoverpa armigera)的致病性,探讨棉铃虫作为Lm感染模型的可行性。用野生株EGDe(中毒)、PrfA缺失株(EGDeΔprfA,弱毒)和PrfA组成型高表达株(EGDeΔprfA+pERL3-prfA~*,高毒)腹腔微量注射4龄棉铃虫幼虫,计算半数致死量(LD_(50))和虫体的载菌量,同时观测棉铃虫中肠细胞的病理变化以及血细胞包囊作用和数目。实验结果显示:①EGDe、EGDeΔprfA和EGDeΔprfA+pERL3-prfA~*的LD_(50)分别为2.56×10~3 cfu/mL、1.98×10~6 cfu/mL和6.63×10~2 cfu/mL。②感染Lm后,EGDe和EGDeΔprfA+pERL3-prfA~*在棉铃虫体内各部分的活菌数均有所上升,而EGDeΔprfA却显著下降。③中肠病理切片显示,Lm对棉铃虫幼虫中肠细胞的损伤程度与菌株的毒性高低正相关,并且随时间延长差异更为明显。④EGDeΔprfA+pERL3-prfA~*对棉铃虫血细胞的包囊作用的抑制作用最强,而EGDeΔprfA基本无抑制作用。⑤感染Lm后,棉铃虫的血细胞数量先急剧上升随后减少。72 h时,感染EGDe、EGDeΔprfA和EGDeΔprfA+pERL3-prfA~*的血细胞减少量分别为45%、27%和71%。结果表明,Lm不仅能成功侵染棉铃虫幼虫并致其死亡,且其半数致死量、其对棉铃虫中肠细胞的损伤程度以及对血细胞包囊作用和数量的影响等均与细菌毒力高低有较好的关联性,表明棉铃虫幼虫(至少是4龄棉铃虫幼虫)适合作为研究Lm致病机制的昆虫模型。     

单核细胞增生李斯特氏菌PCR-DHPLC检测新技术的建立

应用PCR结合变性高效液相色谱(DHPLC)技术建立食品中单核细胞增生李斯特氏菌fimY的快速检测方法。根据单核细胞增生李斯特氏菌prfA和hlyA基因序列的特点设计特异性引物,PCR扩增的产物经DHPLC技术进行快速检测。以单核细胞增生李斯特氏菌等61株参考菌株做特异性试验;单核细胞增生李斯特氏菌菌株稀释成不同梯度,进行灵敏度试验。试验结果表明该方法具有很好的特异性,灵敏度较高,检测低限可达到为181CFU/ml。可以快速、准确检测单核细胞增生李斯特氏菌,是食品中致病菌快速检测的新技术。     

VirAB在单核细胞增生李斯特菌耐药性及生物被膜形成中的作用

【背景】单核细胞增生李斯特菌(Listeria monocytogenes,Lm)对一些临床常用抗生素、乳酸链球菌素(Nisin)等抗菌药物的敏感性下降,然而其背后的机制仍未完全阐明。【目的】调查转运蛋白VirAB在Lm对抗菌药物的耐药性及生物被膜形成中的作用。【方法】利用同源重组技术构建Lm基因缺失突变株,比较野生株和缺失株对抗菌药物的耐药性;利用微孔板法观测突变株生物被膜形成能力的变化;利用平板泳动法研究菌株的泳动能力。【结果】与野生株相比,virAB缺失突变株对头孢类抗生素、Nisin和溴化乙锭的敏感性增加;当培养基中分别添加亚致死浓度的苯扎氯铵、卡那霉素和四环素时,突变株均表现出不同程度的生长缺陷。缺失virAB后菌株形成生物被膜的能力下降。【结论】VirAB在Lm对头孢类等抗菌药物的耐药及生物被膜形成方面具有重要作用。     

单核细胞增生李斯特菌抗酸应激机制

单核细胞增生李斯特菌(Listeria monocytogenes,简称单增李斯特菌)是重要的人畜共患食源性病原,在青贮饲料、发酵食品、宿主胃内以及巨噬细胞吞噬体内都会遭遇酸应激。该菌有多种抗酸应激系统,如F0F1-ATPase、谷氨酸脱羧酶(Glutamate decarboxylase system,GAD)、精氨酸脱亚胺酶(Arginine deiminase,ADI)、鲱氨酸脱亚胺酶(Agmatine deiminase,Ag DI)系统等。在环境pH(pHex)4.5条件下可维持其细胞内pH(pHi)稳态,在pHex 3.5时仍能存活;用温和酸应激(pHex 4.5)预处理单增李斯特菌,可以通过酸耐受反应(Acid tolerance response)提高其在致死性酸性环境中的存活率,这一过程受σB正调控,即σB激活可以保护单增李斯特菌应对多种环境应激。因此,σB可以作为新型抗菌药物的靶标。更为重要的是,弱酸性发酵食品要严格控制李斯特菌的污染,以降低消费者的感染风险。     

单核细胞增生李斯特菌单克隆抗体的制备与鉴定

以单核细胞增生李斯特菌细胞碎片免疫BALB/c小鼠,间接ELISA法成功筛选获得2株稳定分泌抗LM的单克隆杂交瘤细胞株4A7、4H11.抗体效价为1∶160 000以及1∶20 000,亚型为IgG1、IgG2a,Dot-ELISA结果表明4A7和4H11单克隆抗体具有很好的属特异性,Western blot分析表明4A7、4H11抗体分别与单核细胞增生李斯特菌62 kDa以及32 kDa外膜蛋白抗原表位结合,胶体金免疫电镜实验进一步确证以上抗体可有效识别单核细胞增生李斯特菌细胞表面抗原.     

单核细胞增生李斯特菌中RsbU缺失株的构建及对生物被膜形成的影响

单核细胞增生李斯特菌(Listeria monocytogenes,Lm)是一种重要的革兰氏阳性食源性致病菌,它能在大多数活性或非活性固体表面形成生物被膜,从而使抗逆性大大增强并且难以清除,给食品行业造成很大困扰。Sig B(σB)作为革兰氏阳性菌中主要的压力应答因子,在Lm生物被膜形成中起着重要作用,而Rsb U是单核细胞增生李斯特菌Sig B操纵子中的主要信号(能量和物理化学信号)传导蛋白。为检测Rsb U在Lm生物被膜形成中的作用以及与Sig B的关系,本实验构建了rsb U和sig B基因单缺失及双缺失突变株,比较在不同温度(25℃和37℃)和营养环境(营养丰富的BHI培养基和营养贫乏的MEM基础培养基)下,野生株和突变株生物被膜形成能力的差异。结果表明,缺失Rsb U和Sig B显著降低Lm在不同温度和培养基中生物被膜的形成能力;低温(25℃)和贫瘠的营养条件(MEM)更有利于Rsb U传递压力信号激活Sig B,从而作用Lm生物被膜的形成。     

单核细胞增生李斯特菌的检测技术进展

单核细胞增生李斯特菌（Listeria monocytogenes）是一类人畜共患的食源性致病菌。近年来其检测技术取得了迅猛的发展,本文对目前使用的基于培养、免疫学和分子生物学技术的三大类单核细胞增生李斯特菌检测方法进行了综述,同时对单核细胞增生李斯特菌检测的新策略进行了展望。     

Bioluminescent Listeria monocytogenes provide a rapid assay for measuring biocide efficacy

Abstract A recombinant derivative of Listeria monocytogenes 23074, engineered to express the luxAB genes of Vibrio fischeri MJ1, has a bioluminescent phenotype that provides a rapid monitor of microbial viability. The antibacterial activity of phenol and chlorhexidine diacetate (Hibitane) was measured using both bioluminescence and viable counts. Concentration exponents were assessed as 7.3 for phenol and 2.63 for chlorhexidine diacetate using plate counts. The rapidity of bioluminescence measurement constitutes a major advantage in biocide assessment.     

原核表达MurA蛋白并制备其多克隆抗体用于免疫磁珠快速检测单增李斯特菌

【目的】制备MurA多抗,结合免疫磁珠与选择平板进行单增李斯特菌的快速检测,建立单增李斯特菌的免疫磁珠快速检测方法。【方法】构建MurA的原核表达载体,转化大肠杆菌进行优化表达。镍柱纯化表达产物,质谱鉴定重组蛋白,再免疫小鼠,制备其多克隆抗体。用所获多抗制备免疫磁珠,建立单增李斯特菌免疫磁珠-选择性培养基检测方法,并对人工污染牛奶样品进行检测。【结果】在大肠杆菌中高效表达了分子量约为72 kD的可溶性融合蛋白,质谱鉴定其为MurA蛋白;免疫小鼠获得的抗血清效价达1:10 000,与伤寒沙门氏菌、副溶血弧菌、大肠杆菌及属内其它病原菌均无交叉;所建立的免疫磁珠-选择性培养基检测法可检出浓度为103 CFU/mL及以上的单增李斯特菌,仅与英诺克李斯特菌存在一定交叉反应;牛奶样品单次仅需9 h增菌就能被检出,较常规增菌时间缩短39 h;检测限为0.4 CFU/mL。【结论】表达并纯化得到高纯度的单增李斯特菌MurA蛋白,制备的鼠源多克隆抗体亲和力高,特异性好;建立了快速检测单增李斯特菌的免疫磁珠联合选择性培养基法,在灵敏度不变的情况下,实现24 h内成功对牛奶样品的检测,较国标法减少42 h以上。     

Luminol-enhanced chemiluminescence by Listeria monocytogenes: A test for rapid assessment of antimicrobial agents

Abstract Listeria monocytogenes produces chemiluminescence in brain heart infusion broth at 37°C in the presence of carbonate ions and acetaldehyde. This phenomenon can be enhanced by the use of luminol rather than acetaldehyde. Furthermore, there is direct relationship between the extent of growth and the level of luminescence which culminates at the end of the exponential growth. This property was used to study the susceptibility of this bacterium to two antiseptics, cetrimonium bromide and chlorhexidine, and to two antibiotics, ampicillin and chloramphenicol. Inhibition of chemiluminescence was proportional to the antimicrobial agents' concentrations and was complete at their minimal inhibitory concentrations.     

毒力基因调控蛋白PrfA促进单核细胞增生李斯特菌生物被膜的形成

单核细胞增生李斯特菌(Listeria monocytogenes,LM)是重要的革兰氏阳性食源性致病菌,易在食品以及各种食品加工、运输和保藏设备的接触面形成生物被膜,从而具有更强的抗逆性而难以彻底清除,因此成为食品卫生安全的重要隐患.PrfA是LM毒力基因转录表达的重要调控因子,通过比较研究LM野生株(EGD和EGDe)、PrfA缺失株(EGDAprfA和EGDeAprfA)、无害李斯特菌(Listeria innocua,LI),携带组成性表达PrfA蛋白的重组无害李斯特菌(LI-pERL3-prfA*)以及重组单核细胞增生李斯特菌(EGDeΔprfA-pERL3-prfA*)生物被膜形成能力的差异,探讨LM重要的毒力调控蛋白PrfA对生物被膜形成的影响.实验结果显示:LM野生株具有较强的生物被膜形成能力,而LI形成生物被膜的能力最弱;PrfA的缺失能降低LM生物被膜的形成能力;组成性高量表达PrfA蛋白可以回复EGDeΔprfA的生物被膜形成能力,但对LI没有增强作用.以上实验结果表明:PrfA在LM生物被膜形成中具有重要的促进作用.     

Listeria monocytogenes as a probe of immune function.

For almost half a century, the mouse model of Listeria monocytogenes infection has been used to analyse both innate and adaptive components of immunity and to discover key immune genes. Vast accumulated knowledge about the disease in mice provides a unique framework for identifying and characterising immune molecules using a variety of experimental approaches. To illustrate the range of questions that can be addressed using modern genetics and genomics tools, the authors provide an overview of the analysis of components of immune signalling networks using the mouse model of L. monocytogenes infection.     

李斯特菌生物被膜形成的调控

单核细胞增生李斯特菌(Listeria monocyiogenes)能引起人和动物脑膜炎、败血症、流产和单核细胞增多等症状,临床发病率在美国和欧洲等西方发达国家大约为2-8例/10万人,死亡率20％-30％或更高,被WHO列为关系食品卫生安全的重要病源细菌之一一[1-2].该菌能在多数固体表面形成生物被膜,在食品生产、加工、运输和保藏过程中,一旦发生细菌感染并形成生物被膜便难以将其彻底清除,严重威胁着食品卫生安全[3],但其生物被膜形成的具体分子机制尚不清楚[4].     

A multidrug efflux transporter in Listeria monocytogenes

A chromosomal gene (mdrL) was found in Listeria monocytogenes L028, showing a high degree of similarity with multidrug efflux transporters of the major facilitator superfamily (family 2). An allele-substituted mutant of this gene failed to pump out ethidium bromide and presented lower minimal inhibitory concentrations of macrolides, cefotaxime and heavy metals. This is the first multidrug efflux pump described in Listeria.     

Utilization of transferrin-bound iron by Listeria monocytogenes

Abstract It has been demonstrated that under iron-restricted conditions, Listeria monocytogenes can utilize iron-loaded transferrin (Tf) from a range of species as its sole source of iron for growth. Human transferrin conjugated to horseradish-peroxidase (HRP-Tf) bound directly to whole cells of L. monocytogenes . This binding was blocked by apotransferrin indicating that the receptor can bind transferrin in either the iron-bound or iron-free form. Transferrin-binding was not host specific because both bovine and equine transferrin inhibited the binding of HRP-conjugated human transferrin. SDS-PAGE and Western blotting of bacterial surface extracts revealed the presence of a transferrin-binding protein of approximately 126 kDa.     

An iron-dependent mutant of Listeria monocytogenes of attenuated virulence

Abstract A bank of Tn 917 -insertional mutants from the facultative intracellular pathogen Listeria monocytogenes was screened by an original method based on bacterial growth on synthetic medium under iron-limiting conditions. One mutant, whose in vitro growth in synthetic medium was specifically dependent upon the availability of iron in its environment, was isolated and characterized. The insertional event occurred in a non-coding region, upstream of a rrn operon and located within a 1100-kb Not I fragment of the physical map, where the virulence genes already identified in L. monocytogenes were also present. Protein analysis by SDS-PAGE revealed a pleiotropic effect of the insertional event on cell-associated proteins, suggesting a polar effect of the transposon on adjacent unknown gene(s). The virulence in the mouse of this mutant was strongly impaired, although it was capable in vitro of growing intracellularly and of spreading from cell to cell, as shown by the production of lytic plaques on cell culture.     

PCR detection of Listeria monocytogenes: a study of multiple factors affecting sensitivity

AIMS: To test, under comparable conditions, several parameters affecting sensitivity of PCR detection in order to establish a PCR procedure suitable for the routine detection of Listeria monocytogenes in food. METHODS AND RESULTS: Beef samples artificially inoculated were used to determine sensitivity of PCR detection under different parameters. As few as 1 CFU g(-1) were detected by DNA extraction using a DNeasy Tissue Kit (Qiagen GmhH, Hilden, Germany) of 1 ml aliquot and PCR amplification with primers directed to the hlyA gene. This PCR protocol was applied in 60 naturally contaminated foods, comparing two enrichment procedures with the traditional culture method. The highest number of positives was recorded by PCR following a 24-h pre-enrichment step at 30 degrees C and a 24-h enrichment step at 37 degrees C. Afterwards, it was applied in 217 naturally contaminated foods and 56 of them tested positive for L. monocytogenes in which only 17 tested positive using the culture method. CONCLUSIONS: The PCR procedure described has proved to be a rapid and sensitive method suitable for the routine analysis of different types of food. SIGNIFICANCE AND IMPACT OF THE STUDY: The method proposed for the detection of L. monocytogenes, has been validated in naturally contaminated food and is suitable to implement in the food industry.