食源性单增李斯特菌与英诺克李斯特菌基因组特征比较

目的 通过全基因组测序对甘肃省市售食品中分离的单增李斯特菌和英诺克李斯特菌基因组特征进行比较分析。方法 收集2021—2022年甘肃省市售食品中分离的25株单增李斯特菌和7株英诺克李斯特菌作为研究对象,对菌株进行全基因组测序,分析其系统发育谱系、克隆复合群（CC）、序列型（ST）、毒力基因、抗性基因及泛基因组。结果 32株李斯特菌分属单增李斯特菌谱系Ⅰ和Ⅱ及英诺克李斯特菌3个群,单增李斯特菌分为10个亚群,英诺克李斯特菌分为5个亚群,与CC型保持一致,核心基因组多位点序列分型能将各谱系中不同CC型的菌株明显分开,谱系Ⅰ与英诺克李斯特菌的进化关系更近。25株单增李斯特菌均携带李斯特菌毒力岛LIPI-1和内化素基因,不携带LIPI-3,有2株ST87型菌株携带LIPI-4;7株英诺克李斯特菌均不携带LIPI-1和内化素基因,均携带LIPI-4,有5株菌携带LIPI-3。单增李斯特菌有16株携带SSI-1、3株携带SSI-2,7株英诺克李斯特菌均不携带SSI-1,有6株携带SSI-2。李斯特菌的泛基因组大小随着测序基因组数目的增加呈现线性增多,25株单增李斯特菌当菌株数量达到15后核心基因数目稳定在2 272个,占泛基因组基因数目的46.2%,25株单增李斯特菌和7株英诺克李斯特菌共同的核心基因1 487个,当菌株数量达到10后数目趋于稳定。结论 核心基因组多位点序列分型可将不同谱系不同克隆复合群的李斯特菌进行区分,英诺克李斯特菌与单增李斯特菌生化特性相似与其亲缘关系相近有关,致病性差异与英诺克李斯特菌缺失单增李斯特菌特有的毒力基因相关。     

荧光免疫层析法结合免疫磁珠分离技术快速检测单增李斯特菌

为建立单增李斯特菌简单快速、灵敏度高和特异性强的检测方法,本研究以抗单增李斯特菌单克隆抗体偶联磁珠制备免疫磁珠;以羧基荧光微球标记的抗单增李斯特菌多克隆抗体及鼠IgG为标记抗体,抗单增李斯特菌多克隆抗体和羊抗鼠二抗分别作为检测线和质控线制备荧光免疫层析试纸条。将免疫磁珠分离与荧光免疫层析法相结合应用于单增李斯特菌的现场快速检测中。结果表明:荧光免疫层析试纸条对纯培养单增李斯特菌的检测限为4×105CFU/mL,联合检测方法10倍、100倍浓缩时,检测限分别为4×104CFU/mL和1×104CFU/mL。联合检测体系特异性较好,与实验室保存的10株细菌无交叉反应。人工污染样本检测限为1×104CFU/mL,同纯培养物相比检测灵敏度并没有降低。本方法的建立对于食品中单增李斯特菌的现场快速检测具有重要意义。     

恒温实时荧光法快速检测不同样品中的单增李斯特菌

为实现乳制品中、临床病人腹泻物中单增李斯特菌的快速检测,选用外引物F3和B3、内引物FIP和BIP作为环介导等温扩增(loop-mediated isothermal amplification,LAMP)的特异性引物,采用便携式荧光恒温扩增仪作为检测平台,选取单增李斯特菌标准菌株进行基因组DNA灵敏度和最低检测限测定;选用人工污染的15份乳制品样品和20份腹泻样品进行适用性实验;以上样品同时利用国标法GB4789.30-2010进行菌落计数。结果表明:恒温实时荧光法对纯培养的单增李斯特菌基因组DNA、乳制品和腹泻样品中的单增李斯特菌基因组DNA灵敏度均达到10~2CFU/mL、检测限均达到10~3 CFU/mL;阴性样本出现假阳性的概率分别为0、0.03%和0;单增李斯特菌污染浓度在检出限以上的阳性样本检出率分别为100%、99%和92%。研究表明恒温实时荧光法的检测结果与传统国标培养结果基本一致,恒温实时荧光法适用于乳制品中和临床腹泻样本中单增李斯特菌的快速检测。     

添加扩增内标的单增李斯特菌PCR检测方法的建立与应用

以单增李斯特菌inl A基因为靶基因,设计一对特异性引物,以16S rRNA为扩增内标对照,建立了一种含有扩增内标的单增李斯特菌PCR检测方法。优化了PCR反应体系,并对PCR检测方法的特异性、灵敏度、人工污染样品及食品样品检测效果进行了测试。对2株单增李斯特菌、1株英诺克李斯特菌以及19株非李斯特菌菌株进行PCR检测,结果显示,只有2株单增李斯特菌能被检出大小为826 bp的特异性片段,其余20株细菌只能检出1 500 bp的扩增内标片段。灵敏度实验结果显示,基因组DNA和纯培养物的最低检出限分别为1.80×10~2fg/μL和1.21×10~2CFU/m L。当人工污染牛乳样品中单增李斯特菌在最低接种量为0.48 CFU/m L时,经8 h增菌培养后可被该方法检出。采用该研究建立的检测方法对36种食品样品进行检测,证实了该检测方法可以指示检测过程中出现的假阴性现象。综上所述,该研究建立的PCR检测方法能特异性的检测单增李斯特菌,并可有效排除检测过程中出现的假阴性现象,提高检测的准确性。     

单增李斯特菌检测技术研究进展

单增李斯特菌(Listeria monocytogenes)是一种人畜共患食源性致病菌,可使人畜患脑膜炎、心肌炎、败血症、早产等疾病,危害较大。有效控制食品中的单增李斯特菌,是食品安全的重要课题之一。本文对单增李斯特菌的主要检测技术,如传统分离鉴定、免疫法、分子生物学法、全自动微生物分析系统、生物传感器检测作了简要叙述,为深入研究提供参考。并对我国单增李斯特菌的检测监控进行了展望。     

血啉甲醚对单增李斯特菌的光动力灭活作用及机理

通过平板菌落计数法研究血啉甲醚对单增李斯特菌的光动力灭活作用,同时采用聚丙烯酰胺凝胶电泳和聚合酶链式反应分析光动力灭菌技术对单增李斯特菌蛋白降解效果和基因组DNA的损伤程度。实验发现浓度为25μg/mL的血啉甲醚在光功密度为200 mW/cm2的溴钨灯光照射30 m in杀灭了99.9999%的单增李斯特菌,并导致了其蛋白质降解和基因组DNA片段断裂。血啉甲醚对单增李斯特菌的光动力灭活作用非常显著,其灭活机理可能是通过对蛋白质降解和基因组DNA损伤实现的。     

成都市冷藏即食预包装熟肉制品中单核细胞增生李斯特菌定量检测及污染风险研究

目的 为探明了解冷藏即食预包装熟肉制品中单核细胞增生李斯特菌（单增李斯特菌）污染情况及潜在风险,本研究针对短保质期的冷藏即食预包装熟肉制品开展单增李斯特菌定量污染风险调查。方法 2022年5~8月,从成都市采集样品64份,依据采用传统培养分离鉴定和RT-PCR分子检测方法进行单增李斯特菌的定性和定量检测,分离菌株提取DNA后进行二代全基因组测序并开展相关流行病学特征分析。结果 检测结果发现64份即食熟肉样品中,6份样品检出单增李斯特菌,检出份数为6/64。6份阳性样品均由某同一工厂生产,其中5份为卤肉三拼样品。定量检测结果表明,卤肉三拼的单增李斯特菌污染水平为30~200 CFU/g;单增李斯特菌阳性的猪肉样品污染水平为<10 CFU/g。阳性样品中的6株分离菌株经全基因组测序分析鉴定为单增李斯特菌,均含有LIPI-1毒力岛相关基因,多序列位点分型发现主要的序列型（ST）为ST3（n=2）和ST121（n=2）,其次是ST8和ST9。单核苷酸多态性（SNP）结果分析发现同一工厂分离到的单增李斯特菌菌株LM22071911和LM22080802之间检测到23个SNP差异,单增李斯特菌株LM22062706和LM22080806之间检测到13个SNP差异,说明在该工厂加工环节存在两种克隆群的单增李斯特菌直接传播的风险。结论 成都市部分市售冷藏即食预包装熟肉制品污染多种李斯特菌,需加强源头控制。     

多重荧光定量PCR法检测海产品中副溶血性弧菌、沙门菌和单增李斯特菌

目的建立检测海产品中副溶血性弧菌、沙门菌和单增李斯特菌的多重荧光定量PCR体系。方法针对副溶血性弧菌tlh基因,沙门菌Ompc基因和单增李斯特菌hly基因设计引物和Taq Man探针,建立多重荧光定量PCR体系,进行特异性与敏感性研究;利用该体系检测海产品中的副溶血性弧菌、沙门菌和单增李斯特菌。结果副溶血性弧菌、沙门菌和单增李斯特菌可得到特异性扩增,而共存于海产品中的其他细菌均未见扩增曲线。敏感性试验显示,该体系对副溶血性弧菌、沙门菌和单增李斯特菌的最低检测限分别为72、40、80 cfu/ml。对舟山采集的150份样品进行检测,检出32份副溶血性弧菌、11份沙门菌、5份单增李斯特菌,与国标法检测结果一致。结论本研究建立的基于Taq Man探针的多重荧光定量PCR检测方法可以特异、灵敏、简单快速地实现对海产品中副溶血性弧菌、沙门菌和单增李斯特菌的检测。     

猪肉制品中单增李斯特菌污染现状及致病力控制分析

近年来，由单增李斯特菌污染猪肉制品引起的食品安全事故频发，对猪肉制品的食用安全造成严重威胁。加强对猪肉制品中单增李斯特菌致病力的控制可有效遏制社会公共卫生问题的发生。文章首先归纳了近些年猪肉中单增李斯特菌的污染现状，然后综述了单增李斯特菌的致病能力，最后从生长特性、抗性和毒性3个方面剖析了控制单增李斯特菌致病力的可行性，并且提出了可用“栅栏技术”从不同方面控制单增李斯特菌，从而提高猪肉的微生物安全。     

不同季节原料乳中主要微生物和理化指标分析

研究了冬季、春季和夏季原料乳的主要理化指标和微生物指标。3个季节内,蛋白质、乳脂、乳糖和干物质的变化范围分别是3.38%～3.52%,3.98%～4.26%,4.80%～4.85%和12.78%～13.19%。理化指标检测结果表明冬季牛乳的营养成分高于春夏两季,并且3个季节的乳成分均高于生鲜牛乳的收购标准;此外,对原料乳中主要的微生物:总菌数、乳酸菌、大肠菌群、沙门氏菌、蜡样芽孢杆菌、单增李斯特菌和嗜冷菌的菌数进行了检测。结果中未检测到沙门氏菌、蜡样芽孢杆菌和单增李斯特菌,其他微生物质量分数均在可接受范围内,但是大肠菌群的出现说明需要建立相关的卫生质量标准。     

Optimizing detection of heat-injured Listeria monocytogenes in pasteurized milk.

Optimal conditions for the detection of heat-injured cells of Listeria monocytogenes in modified Pennsylvania State University (mPSU) broth were determined using a response surface design generated by a computer program, EChip. Different combinations of incubation temperatures and lithium, magnesium, and D-serine concentrations were evaluated to determine the optimum conditions for the detection of heat-injured L. monocytogenes in filter-sterilized whole milk inoculated with selected problematic background microflora. A concentration of 212 mM lithium chloride completely inhibited the growth of Enterococcus faecium while permitting recovery and detection of L. monocytogenes. A concentration of 15.8 mM MgSO4 was found to be optimum for the recovery and detection of L. monocytogenes. A concentration of 140.2 mM D-serine was found to completely inhibit the germination of Bacillus subtilis var. globii spores but not recovery and detection of L. monocytogenes. Under optimum concentrations of LiCl, MgSO4, and D-serine and in the absence of background microflora, the effect of incubation temperature on percentage detection was described by a second-order polynomial model, and 28 degrees C was determined to be optimal. In the presence of background microflora, the effect of incubation temperature on percentage detection of heat-injured cells was described by a third-order polynomial model, and 30 degrees C was found to be optimal. Optimizing the levels of highly specific and selective agents, nutrients, and incubation temperature in one recovery enrichment system dramatically increased the Listeria/background microflora ratio. This resulting medium, optimized PSU (oPSU) broth, greatly improved the detection of heat-injured and nonheat-injured L. monocytogenes by both conventional and molecular methods (Oxoid's Listeria Rapid Test, Gen-Probe's Accuprobe Listeria monocytogenes Culture Identification Test, and Qualicon's BAX for screening Listeria monocytogenes).     

Evaluation and interlaboratory validation of a selective agar for phosphatidylinositol-specific phospholipase C activity using a chromogenic substrate to detect Listeria monocytogenes from foods

Phosphatidylinositol-specific phospholipase C (PI-PLC) activity is a potential virulence factor and is exhibited only by the Listeria species Listeria monocytogenes and Listeria ivanovii. A chromogenic substrate for the direct detection of PI-PLC activity is available in a new medium (BCM L. monocytogenes plating agar). The use of a chromogenic substrate offers a mechanism with which to directly screen for L. monocytogenes and L. ivanovii other than the esculin used in Oxford (OXF) and Palcam (PAL) agars, which screen for all Listeria species. The specificity levels of BCM plating agar and of BCM confirmation and rhamnose agars were evaluated with 107 Listeria and 10 Bacillus species isolates. In addition, BCM L. monocytogenes plating agar was compared with standard Listeria selective agars (OXF and PAL agars) with regard to the recovery of L. monocytogenes from 2,000 food and environmental samples obtained from eight participating laboratories. A Listeria species was isolated from at least one of the agars in 209 analyses, and L. monocytogenes was isolated in 135 of these analyses. In 27 of the analyses in which L. monocytogenes was isolated, one or more of the selective differential agars used failed to isolate L. monocytogenes, and therefore the results of these analyses were discrepant. Relative to a reference method involving the use of all three agars (OXF, PAL, and BCM agars), the OXF-BCM, PAL-BCM, and OXF-PAL combinations had sensitivities of 99.3, 99.2, and 90.2%, respectively. In statistical analyses of the different combinations of agars, the OXF-BCM and BCM-PAL combinations were found to be superior to the OXF-PAL combination for the detection of L. monocytogenes.     

单核细胞增生李斯特氏菌实时荧光SPIA方法的建立

以单核细胞增生李斯特氏菌（Listeria monocytogenes,L.monocytogenes）hly A基因为靶序列,设计5’端为RNA序列,3’端为DNA序列的嵌合引物和链终止Blocker序列,经过优化,建立实时荧光单引物等温扩增（Real-time fluorescence SPIA）方法,进行特异性、检出限实验,并对不同DNA提取方法进行比较。结果显示,确定荧光染料SPIA法的最佳反应温度为58℃,反应30 min,引物特异性良好,只有4株不同来源的L.monocytogenes DNA产生典型的S型荧光扩增曲线。对L.monocytogenes纯培养DNA的检出限为3.6×10¹fg/μL,相应菌液浓度为1.2×10¹CFU/m L;Realtime fluorescence SPIA检测试剂盒法、水煮法、溶菌酶-蛋白酶K法、饱和酚提取法四种方法提取DNA,均产生典型的扩增曲线,Ct值没有明显差异。对人工添加猪肉火腿样品中的L.monocytogenes,采用水煮法提取DNA的检出限为1.1×10²CFU/g。结果表明,所建立的L.monocytogenes的Real-time fluorescence SPIA新型等温扩增方法,特异性强、灵敏度高、不受DNA纯度的影响、快速、简便。      

单核细胞增生性李斯特氏菌污染及其检测方法研究进展

单核细胞增生李斯特菌是一种能引起人畜共患的食源性致病菌之一。近年来,在许多国家,它被列为卫生部门重点检测的几种食源性致病菌之一。主要介绍了单核细胞增生李斯特菌的污染及其检测方法研究进展。     

食品中亚致死损伤单增李斯特菌的研究进展

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。食品加工过程中所产生的亚致死损伤单增李斯特菌是不容忽视的,在适宜的环境下,损伤菌会恢复至正常状态继续生长,对消费者的健康造成威胁,因此亚致死损伤菌的存在是食品安全的一大隐患。探究亚致死损伤菌的检测、修复是目前研究的热点之一,本文将综合相关研究对近年来食品中亚致死损伤单增李斯特菌的检测、修复方法以及发展趋势进行综述。     

Efficiency of four secondary enrichment protocols in differentiation and isolation of Listeria spp. and Listeria monocytogenes from smoked fish processing chains

Four secondary enrichment protocols (conventional methods: UVM II, Fraser 24 h and Fraser 48 h: Impedimetric method: Listeria electrical detection medium) were studied for their ability to isolate Listeria spp. and Listeria monocytogenes from fish and environmental samples collected along the processing chain of cold-smoked fish. From all methods, Listeria spp. and L. monocytogenes were respectively present in 56 and 34 of 315 samples analysed. Fraser broth incubated for 48 h gave the fewest false negative Listeria spp. results [4/56; (7.1%)], but concurrently only 15/34 (44.1%) samples were correctly identified as containing L. monocytogenes, Listeria electrical detection (LED) medium detected only 36/56 (64.3%) Listeria spp. positive samples. Despite this lower isolation rate, LED identified 20/34 (58.8%) L. monocytogenes positive samples correctly and gave fewer false positive results. The overall conclusion was that more than one isolation method is needed to accurately estimate L. monocytogenes contamination rates.     

A rapid polymerase chain reaction (PCR)-based assay for the identification of Listeria monocytogenes in food samples.

A rapid and simple assay has been developed which allows specific detection of Listeria monocytogenes within 3.5 h in cultures prepared from suspect food samples and propagated 48 h in selective medium. The assay is based on PCR technology, and uses a specific primer set derived from sequences located down-stream of the hlyA gene. The specificity of the primer set was confirmed by testing 115 L. monocytogenes, 14 L. innocua, 5 L. seeligeri and 4 L. ivanovii isolates. The assay was compared to standard microbiological tests and gave identical results for 83 food samples, including 32 positives. These field trials indicate that the assay developed provides an alternative detection system for L. monocytogenes in foods, which can be used by the food industry.     

Molecular Subtyping and Tracking of Listeria monocytogenes in Latin-Style Fresh-Cheese Processing Plants

Latin-style fresh cheeses, which have been linked to at least 2 human listeriosis outbreaks in the United States, are considered to be high-risk foods for Listeria monocytogenes contamination. We evaluated L. monocytogenes contamination patterns in 3 Latin-style fresh-cheese processing plants to gain a better understanding of L. monocytogenes contamination sources in the manufacture of these cheeses. Over a 6-mo period, 246 environmental samples were collected and analyzed for L. monocytogenes using both the Food and Drug Administration (FDA) method and the Biosynth L. monocytogenes detection system (LMDS). Finished cheese samples from the same plants (n = 111) were also analyzed by the FDA method, which was modified to include L. monocytogenes plating medium (LMPM) and the L. monocytogenes confirmatory plating medium (LMCM) used in the LMDS method. Listeria monocytogenes was detected in 6.3% of cheese and 11.0% of environmental samples. Crates, drains, and floor samples showed the highest contamination rates, with 55.6, 30.0, and 20.6% L. monocytogenes positive samples, respectively. Finished products and food contact surfaces were positive in only one plant. The FDA method showed a higher sensitivity than the LMDS method for detection of L. monocytogenes from environmental samples. The addition of LMPM and LMCM media did not further enhance the performance of the FDA method for L. monocytogenes detection from finished products. Molecular subtyping (PCR-based allelic analysis of the virulence genes actA and hly and automated ribotyping) was used to track contamination patterns. Ribotype DUP-1044A, which had previously been linked to a 1998 multistate human listeriosis outbreak in the United States, was the most commonly identified subtype (20/36 isolates) and was isolated from 2 plants. This ribotype was persistent and widespread in one factory, where it was also responsible for the contamination of finished products. We hypothesize that this ribotype may represent a clonal group with a specific ability to persist in food processing environments. While previous listeriosis outbreaks were linked to Latin-style fresh cheeses made from unpasteurized milk, the presence of this organism in pasteurized cheese products illustrates that persistent environmental contamination also represents an important source of finished product contamination.     

食源性单增李斯特菌检测技术研究进展

由单核细胞增生李斯特氏菌(简称单增李斯特菌)(Listeria monocytogenes,L.monocytogenes)引起的李斯特菌病,被认为是世界范围内主要的食源性疾病之一,致死率可高达20％～30％.单增李斯特菌普遍存在于各类食品中,其在低温、有氧或无氧条件下、以及较宽的pH和渗透压范围内均可生长繁殖.因此,...     

免疫磁分离技术在食源性单增李斯特菌检测中应用的研究进展

食源性单增李斯特菌是李斯特菌属中的唯一能引起人类疾病的病原菌,致死率30%~70%,并严重威胁着人类健康。早期快速准确地检测出食品中可能污染的单增李斯特菌对于减少死亡率非常重要,因此亟需建立一些快速、灵敏和高特异性的检测方法。现有单增李斯特菌的检测方法对未经前增菌的食品样本检测灵敏度较低,限制了这些方法直接用于食品样本中单增李斯特菌的快速检测。免疫磁分离是一种可以短时间内高效富集样本中目的菌的技术,与常用的检测方法结合,可以缩短检测周期,提高检测灵敏度。本文综述了免疫磁分离技术在食源性单核细胞增生性李斯特检测中应用的研究进展。