Mini-VIDAS法和国标法检测单增李斯特菌的效果比较

目的比较两类方法检测单增李斯特菌的特异性、灵敏度和抗干扰性,并研究添加成分和样品基质对检测效果的影响。方法选取单增李斯特菌(CICC 21633/ATCC 19111)、斯氏李斯特菌(CICC 21671)、伊氏李斯特菌(CICC 21663/ATCC19119)和英诺克李斯特菌(CICC 10417/ATCC 33090)为试验对象,采用国标法和mini-VIDAS法为试验方法。结果两种检测方法的特异性较好,均能区分李斯特菌属和非李斯特菌属。国标法和mini-VIDAS法的灵敏度分别为104~105 cfu/m L和105 cfu/m L;同时,国标法的抗干扰性稍强,1/104混合干扰菌液可检出目标菌。添加次氯酸钠后两种方法对目标菌的检出都受到影响。添加成分对单增李斯特菌生长的抑制作用符合乙醇苯甲酸钠十三香料凉拌菜料次氯酸钠。当基质中单增李斯特菌污染水平较低并且背景微生物数量高时,目标菌的检测结果受基质严重干扰。不同增菌液对单增李斯特菌的增菌效果为Fraser肉汤LB2FB2。结论 mini-VIDAS法和国标法对单增李斯特菌的检测性能基本相当。采用国标法时需考虑微生物污染背景、添加成分等样品基质特性,以提高目标菌的检出率。mini-VIDAS法可作为初筛的良好工具。     

单增李斯特菌国标检验培养基质量的比较

通过考察市售单增李斯特菌国标检验培养基对单核细胞增生李斯特氏菌(Listeria monocytogenes,LM,以下简称单增李斯特菌)和非单增李斯特菌的增菌检测效果,比较不同品牌培养基的质量。该研究采用螺旋涂布计数法,定量测定30株单增李斯特菌和9株非单增李斯特菌在4个品牌的李氏增菌肉汤(LB₁、LB₂)和6个品牌的PALCAM琼脂培养基、李斯特氏菌显色培养基中的生长情况。实验结果表明:30株单增李斯特菌在4个品牌的LB;增菌肉汤中的浓度均值为2.04×10⁷CFU/m L~4.59×10⁷CFU/m L;在LB;增菌肉汤中的浓度为2.36×10⁷CFU/m L~2.69×10⁷CFU/m L;在D品牌的PALCAM琼脂培养基中的生长率均值为0.55,在其余5个品牌的PALCAM琼脂培养基和6个品牌的李斯特氏菌显色培养基中的生长率均值均超过0.85。30株单增李斯特菌在李氏增菌肉汤(LB₁、LB₂)、PALCAM琼脂培养基、李斯特氏菌显色培养基中的增菌检测效果存在显著性差异(p<0.05)。4个品牌的李氏增菌肉汤的增菌效果和6个品牌PALCAM琼脂培养基、李斯特氏显色培养基的检测效果存在显著性差异(p<0.01)。该研究结果表明市售单增李斯特菌国标检验培养基质量差异大。     

即食菜卷和肉汤中单增李斯特菌生长模型及控制研究

通过对单增李斯特菌在不同NaCl浓度、不同pH、不同温度下营养肉汤中生长情况的研究,确定了单增李斯特菌的最佳生长条件。建立了30℃下单增李斯特菌在营养肉汤和即食菜卷中的生长模型。以模型为依据,提出了即食菜卷加工中控制李斯特菌的措施。     

基于酸处理的产志贺毒素大肠埃希菌选择性增菌方法研究

目的开发一套基于酸处理的产志贺毒素大肠埃希菌(Shiga toxin-producing Escherichia coli,STEC)选择性增菌方法,提高食品中STEC菌株的检出率。方法 选择12株不同血清型STEC菌株以及13株常见干扰菌,评价其对不同酸处理条件(pH2.0、2.5、3.0)的耐受性;考察筛选的酸处理条件对营养胁迫和冷冻胁迫状态STEC菌株生长活性的影响;比较研究酸水解酪蛋白、酵母提取物和丙酮酸钠等成分对胁迫状态STEC菌株的促生长作用,优化酸处理后中和/促生长培养基配方;比较增菌前与增菌后两种酸处理方式对STEC菌株分离效果的影响;最后通过人工污染样品,评价本研究建立的STEC酸处理选择性增菌方法的检测效果。结果 本研究建立了一种不依赖于抑菌成分的STEC选择性增菌方法,样品在增菌前经酸处理培养基室温处理2 h,降低背景杂菌干扰,再通过中和/促生长培养基(TSB-1.5%Tris-0.1%丙酮酸钠)进行目标菌的增菌培养;人工污染试验结果表明,本方法与我国现行GB4789.36-2016标准方法相比,能够有效减少背景杂菌的干扰、获得更高的STEC分离效率。结论 本研究建立的基于酸处理的STEC选择性增菌方法能够有效提高食品中STEC菌株的检测效率和检测准确性。     

免疫磁珠检测食品中单增李斯特菌的研究

免疫磁珠技术因其具有操作简便、高效快速等特点,目前已被广泛地应用于食品的快速分离纯化和检测领域中。本研究建立了单增李斯特菌(Listeria monocytogenes)的免疫磁珠(Immune magnetic beads)检测方法,实现对单增李斯特菌的快速检测。通过对单增李斯特菌免疫磁珠添加量、免疫磁珠与菌液最佳反应时间、免疫磁珠分离单增李斯特菌的敏感性试验、特异性试验的研究,确定了单增李斯特菌抗体添加量50μg/mL、免疫磁珠添加量100μL,免疫磁珠与菌液最佳作用时间为30min。结果表明免疫磁珠法可快速特异地检测出食品中单增李斯特菌,检测灵敏度底限为10-7,相应的细菌浓度为14cfu/mL。该方法反应灵敏度高,特异性强,大大节省了检测时间和费用,可用于食品中单增李斯特菌的快速检测。因此研究食品中单增李斯特菌快速、准确检测方法以预防和控制食品安全事件的发生,具有重要的现实意义。     

2005—2013年河北省即食食品中单增李斯特菌污染及耐药特征研究

研究2005—2013年河北省即食食品中单增李斯特菌污染状况及耐药特征。方法 单增李斯特菌检验参照(GB/T 4789.30),药物敏感性试验应用微量肉汤稀释法,对15种抗生素进行耐药检测。结果 单增李斯特菌总污染率为1.34%,凉拌菜及沙拉(2.62%)、生食水产品(2.51%)的检出率均高于所测其他食品。136株单增李斯特菌对15种抗生素耐药率为13.97%,以氯霉素耐药率最高(7.35%),其次是四环素和复方新诺明(均为4.41%),强力霉素和环丙沙星的耐药率均为2.94%,所有菌株对青霉素、亚胺培南、头孢噻吩、利福平、氨苄西林-舒巴坦酸敏感。结论 河北省即食食品中存在单增李斯特菌污染,分离菌株存在较严重的耐药情况,且耐药性有增长趋势,应加强即食食品的监管以保证食品安全及公众健康。     

单增李斯特氏菌快速检测方法的建立

建立单增李斯特氏菌的快速检测方法.针对单增李斯特氏菌virR基因序列设计特异性引物及探针,建立等温扩增法,并利用免疫金标试纸条对结果进行检测.用10株单增李斯特氏菌、6株李斯特菌属菌株及其他食源性致病菌24株进行特异性试验;通过定量DNA、纯菌液计数进行灵敏度验证.结果表明建立方法具有较好的特异性;增菌液检测灵敏度为102 cfu/test,DNA检测灵敏度为10°pg/test.建立的单增李斯特氏菌的快速检测方法特异性较好、灵敏度高,适合于单增李斯特氏菌快速筛选检测.     

沙门氏菌、金黄色葡萄球菌、志贺氏菌和单增李斯特菌选择性共增菌培养基的研制

目的:研制一种对沙门氏菌(Salmonella)、金黄色葡萄球菌(Staphylococcus aureus)、志贺氏菌(Shigella)和单增李斯特菌(Listeria monocytogens)的选择性共增菌培养基(SSSL培养基)。方法:挑选添加成分进行单因素试验,确定SSSL培养基的成分及配比,采用平板计数法验证SSSL培养基的增菌效果。结果:确立了SSSL培养基配方,目标菌在SSSL增菌培养基中培养8 h后,菌体浓度都达到了10~5～10~6CFU/m L,而且抑制非目标菌的生长。结论:SSSL培养基能用于沙门氏菌、金黄色葡萄球菌、志贺氏菌和单增李斯特菌选择性共增菌,可望与多种检测方法联用,以提高检测率和准确性。     

基于SnO_2纳米花气体传感器快速检测牛奶中的单增李斯特菌

以单增李斯特菌特征性代谢物3-羟基-2-丁酮为检测靶点,水热法合成与之匹配的敏感材料,制备出单增李斯特菌特异检测半导体气体传感器。采用X射线衍射和透射电子显微镜手段对材料的结构和形貌进行表征。并分析传感器对3-羟基-2-丁酮标准气体的气敏性能及其在牛奶中单增李斯特菌的检测效果。结果表明,本研究合成的SnO_2材料为纳米花结构。制备出的单增李斯特菌特异检测半导体气体传感器对3-羟基-2-丁酮灵敏度高、响应恢复时间短、选择性和稳定性好。用于检测单增李斯特菌时,传感器的灵敏度与细菌浓度呈现出良好的线性关系,线性方程为lg(S-1)=0.198 3lg C-0.472 5(R~2=0.990 1)。本方法快速灵敏、操作简便,可用于食品中单增李斯特菌的快速检测。     

食源性单增李斯特氏菌Lm319的毒力基因在四种肉汤培养基中表达分析

为了探明食源性单增李斯特菌的毒力基因在四种不同天然肉汤中的表达情况,了解食源性单增李斯特菌在不同肉类食品中的毒力水平。对单增李斯特菌Lm319中27个毒力基因进行PCR检测,并对Lm319在猪、鸡、牛和羊四种肉汤培养条件下27个毒力基因的表达情况进行RT-PCR检测。结果显示:单增李斯特菌Lm319中含有23个毒力基因;猪肉汤培养基中能表达的毒力基因种类以及基因表达量都是最高的,其次是羊肉肉汤培养基和牛肉肉汤培养基,而鸡肉肉汤培养基中表达的基因最少;同时发现iap、fbp、hpt和bsh这4个毒力基因的表达与prf A基因存在相关性。本研究表明,单增李斯特菌在不同生长环境下其毒力基因表达存在较大差异。     

Comparison of two chromogenic media for the detection of Listeria monocytogenes with the plating media recommended by EN/DIN 11290-1

The efficacy of two selective chromogenic culture media, Agar Listeria Ottaviani and Agosti (ALOA) and RAPID' L. mono for the detection of Listeria monocytogenes in food, was compared with that of an official culture method according to the EN/DIN 11290-01 and -02 protocols [corresponding to the section 35 LMBG (German Food Act) method]. A total of 310 pre-packed ready-to-eat food samples (100 of graved and cold smoked salmon, 130 of different raw and cooked sausages and 80 of delicatessen and mixed salads) were examined. L. monocytogenes was identified in 52 investigated salmon samples. Using two chromogenic media, 50 samples were found positive for L. monocytogenes. Compared to the reference method there were no false-positive results. By the EN/DIN 11290-01 culture procedure after the selective enrichment in Fraser broth 12 out of 130 samples of sausages were positive for L. monocytogenes. These 12 samples were also positive for L. monocytogenes with the chromogenic medium RAPID' L. mono. One sample was false negative with ALOA. Three additional samples were found positive with ALOA and four with RAPID' L. mono. The standard method was inadequate to confirm these samples as positive. Listeria spp. were isolated from 7 samples of mixed salads with both methods. One, 3 and 3 samples were found to contain L. monocytogenes, L. innocua and L. seeligeri, respectively. Both chromogenic media enabled a rapid and specific detection of L. monocytogenes within 24h after enrichment. Visual detection of pathogenic L. monocytogenes and other Listeria spp. was easier on chromogenic media.     

Reduced detectability of Listeria monocytogenes in the presence of Listeria innocua

Recent foodborne crises have demonstrated the importance of monitoring food safety. In terms of microbiological criteria, food safety requires the reliable detection of pathogens such as Listeria monocytogenes along the food chain by appropriate analytical methods. However, indications exist that accompanying Listeria innocua strains suppress the growth of L. monocytogenes during selective enrichment, which may cause reduced or even inhibited detection. To study these effects, the limit of detection of L. monocytogenes was investigated in the presence of L. innocua using the International Organization for Standardization standard method ISO 11290-1 and the VIDAS LDUO system, an automated method based on enzyme-linked fluorescence technology. The challenge was to provide low initial Listeria concentrations at sufficient precision to quantify the influence on the probability of detection of L. monocytogenes. The application of reference materials appropriate for quantitative test methods and a standardized dilution procedure were necessary to ensure accurate CFU levels of defined proportions of mixtures of both Listeria species. During selective enrichment, overgrowth of L. monocytogenes by L. innocua could be confirmed, leading to high rates of false-negative results. Moreover, with both methods, a significant decrease in the detectability of L. monocytogenes could be quantified at ratios of 2:1 at very low concentrations representative of natural contamination levels often found in foods and environments. It is concluded that there is a need to improve existing procedures with respect to selective enrichment, as well as the detection techniques.     

Liquid and solid selective differential media for the detection and enumeration of L. monocytogenes and other Listeria spp

A selective and differential medium (PALCAM agar) was elaborated for the isolation and enumeration of Listeria monocytogenes. PALCAM is based on Columbia agar with 0.05% glucose made selective by the addition of 0.001% polymyxin B, 0.0005% acriflavin, 1.5% lithium chloride and 0.002% ceftazidime. The diagnostic traits were attained by the incorporation of (i) 0.08% aesculin and 0.05% ferric salt; and (ii) 1% mannitol plus 0.008% phenol red. PALCAM recovered test strains of L. monocytogenes and other Listeria spp quantitatively and suppressed most other bacteria of common occurrence in fresh food. L. monocytogenes colonies were approximately 2 mm grey-green with a black sunken centre and a black halo on a cherry-red background. The occasional Enterococcus or Staphylococcus strains developing on the medium gave rise to grey colonies with a brown-green halo or yellow colonies with a yellow halo. PALCAM was the preferred medium out of 13 tested Listeria selective agars in current use. A similar differential enrichment broth, L-PALCAMY was developed based on peptone yeast extract broth with 2.5% egg yolk emulsion. The diagnostic traits and inhibitors used in this medium were the same as in PALCAM agar, through in different concentrations. Growth rate and cellcrop of L. monocytogenes in L-PALCAMY were of the same order as in Columbia broth. The growth of the majority of other bacteria of common occurrence in fresh foods was inhibited. The medium recovered L. monocytogenes more effectively from severely contaminated food than other current enrichment media.     

单核细胞增生李斯特菌在蜂产品中的生存及检测

目的:为了保障蜂产品免受致病性单核细胞增生李斯特菌的威胁,对蜂产品中单核细胞增生李斯特菌进行检测。方法:采用培养和分子生物学的检测方法研究蜂产品中致病性单核细胞增生李斯特菌的污染状况。将高浓度的病原菌人工污染到蜂蜜和蜂王浆中,室温存放一定时间后,在选择性培养基上增菌培养;以毒力基因hly和16sRNA基因为靶序列,建立双重PCR检测病原菌的方法;以5’、3’端标记FAM、TAMRA的hly基因探针进行荧光定量PCR检测。结果:单增李斯特菌在蜂蜜中的存活时间为5d,而在蜂王浆中不能存活。对未经增菌的人工污染蜂产品采用溶菌酶+蛋白酶K的方法提取DNA,同时采用本实验中建立的双重PCR和荧光定量PCR方法检测,蜂蜜中单增李斯特菌含量均为102CFU/mL。采用这两种方法可在8h内完成蜂蜜中单核细胞增生李斯特菌的快速检测。结论:单增李斯特菌能够在蜂蜜中存活数天,几乎不继续繁殖,而在蜂王浆中不能存活。     

PURIFICATION OF THE ANTI-LISTERIAL BACTERIOCINLIKE INHIBITORY SUBSTANCE PRODUCED BY ENTEROCOCCUS FAECIUM 108

Extracellular antimicrobial substances produced by certain enterococci inhibit Listeria monocytogenes. Enterococcus faecium 108, a competitive food isolate, produced a heat-stable and protease-sensitive anti -L. monocytogenes bacteriocin-like substance (Ef108) in Listeria selective enrichment broth and other media. Ef108 activity was purified to homogeneity by a four-step procedure including (NH₄)₂SO₄ fractionation, chromatography on anion exchange QSepharose column, and two Superose 12 gel filtration columns. In activities represented by two peaks (Ef108A and Ef108B), 90% of the crude activity was due to peak B. Ef108A is believed to be a variable aggregate of the active moiety in Ef108B. All Listeria spp. and five L. monocytogenes serotypes were inhibited by Ef108B in several media including Listeria enrichment medium. The Ef108 activity may be due to a bacteriocin-like inhibitory substance produced by E. faecium 108 that may suppress the growth and predominance of Listeria monocytogenes during selective enrichment .     

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).     

COMPARISON OF SELECTIVE AND NONSELECTIVE ENRICHMENT MEDIA FOR THE ISOLATION OF LISTERIA SPECIES FROM RETAIL FOODS

Enrichment in a nonselective medium, Buffered Peptone Water (BPW) was compared with selective enrichment in University of Vermont Medium (UVMI and UVMII) for the isolation of Listeria spp. from foods. The selectivity of the 2 types of media for the pathogenic strain, Listeria monocytogenes, was also compared. In total, 221 food samples including beef burgers, ham, turkey, lettuce, broccoli, carrots, coleslaw, salads, fish, and ice cream, were purchased from local retail outlets and examined for the presence of Listeria species and L. monocytogenes using both enrichment media Listeria species were detected in 57 (25.8%) samples using UVM, and 56 (25.3%) using BPW. L. monocytogenes was present in 33(14.9%) samples enriched in UVM and in 29(13.1%) samples enriched in BPW. The advantages and disadvantages of selective and nonselective enrichment for detection of Listeria species from a range of foods are discussed.     

Comparative evaluation of culture- and BAX polymerase chain reaction-based detection methods for Listeria spp. and Listeria monocytogenes in environmental and raw fish samples

Two commercial polymerase chain reaction (PCR)-based Listeria detection systems, the BAX for Screening/Listeria monocytogenes and the BAX for Screening/Genus Listeria, and a culture-based detection system, the Biosynth L. monocytogenes Detection System (LMDS), were evaluated for their ability to detect L. monocytogenes and Listeria spp. in raw ingredients and the processing environment. For detection of L. monocytogenes from raw fish, enrichment was performed in Listeria enrichment broth (LEB), followed by plating on both Oxford agar and LMDS L. monocytogenes plating medium (LMPM). Detection of Listeria and L. monocytogenes from environmental samples was performed using LMDS enrichment medium, followed by plating on both Oxford agar and LMPM. A total of 512 environmental samples and 315 raw fish were taken from two smoked fish processing facilities and screened using these molecular and cultural Listeria detection methods. The BAX for Screening/L monocytogenes was used to screen raw fish and was 84.8% sensitive and 100% specific. The BAX for Screening/Genus Listeria was evaluated on environmental samples and had 94.7% sensitivity and 97.4% specificity. In conjunction with enrichment in LEB, LMPM had a sensitivity and specificity for detection of L. monocytogenes from raw fish of 97.8 and 100%, respectively. Use of LMDS enrichment medium followed by plating on LMPM allowed for sensitivity and specificity rates of 94.8 and 100%, respectively, for detection of L. monocytogenes from environmental samples. We conclude that both the BAX systems and the use of LMPM allow for reliable and rapid detection of Listeria spp. and L. monocytogenes. While the BAX systems provide screening results in about 3 days, the use of LMPM allows for L. monocytogenes isolation in 4 to 5 days.     

Comparison of growth kinetics for healthy and heat-injured Listeria monocytogenes in eight enrichment broths

Detection of Listeria in food products is often limited by performance of enrichment media used to support growth of Listeria to detectable levels. In this study, growth curves were generated using healthy and heat-injured Listeria monocytogenes strain F5069 in three nonselective and five selective enrichment broths. Nonselective enrichment media included the current Food and Drug Administration Bacteriological Analytical Manual Listeria enrichment broth base (BAM), Listeria repair broth (LRB), and Trypticase soy broth. Selective enrichment media included BAM with selective agents and LRB with selective agents, BCM L. monocytogenes preenrichment broth, Fraser broth, and UVM-modified Listeria enrichment broth. The Gompertz equation was used to model the growth of L. monocytogenes. Gompertz parameters were used to calculate exponential growth rate, lag-phase duration (LPD), generation time, maximum population density (MPD), and time required for repair of injured cells. Statistical differences (P < 0.05) in broth performance were noted for LPD and MPD when healthy and injured cells were inoculated into the broths. With the exception of Fraser broth, there were no significant differences in the time required for the repair of injured cells. Results indicate that the distinction between selective and nonselective broths in their ability to grow healthy Listeria and to repair sublethally injured cells is not solely an elementary issue of presence or absence of selective agents.