便携式生物传感器在真菌毒素检测中的应用研究进展

食品在贮藏、运输和加工过程中易受真菌侵染。由真菌代谢所产生的真菌毒素对食品造成了严重的污染,并引发了严峻的食品安全问题。传统的检测方法大多需要大型精密仪器设备、繁琐的操作步骤以及专业技术人员,这在很大程度上限制了这些方法在现场即时检测中的应用。便携式传感器允许用户利用小型化设备实时监测真菌毒素的污染情况,具有操作简便、用户友好、检测速度快、灵敏度高等优点,受到越来越多的科研工作者及社会各界人士的青睐。本文重点对试纸条、个人血糖仪、手持式pH计、气压计、智能手机、微流控芯片以及基于可视化、电化学、等离子体等输出信号的便携式生物传感器在真菌毒素检测中的应用和发展前景进行综述,以期为真菌毒素新型便携式生物传感器的研发提供参考。     

pH响应比色酶联免疫吸附法检测牛奶中猪霍乱沙门氏菌

人类常常因误食被猪霍乱沙门氏菌感染的食物而引发中毒。因此,急需构建适用于快速、灵敏检测食物中猪霍乱沙门氏菌污染的新方法。鉴于此,本文构建了一种pH响应比色酶联免疫吸附法灵敏检测牛奶中猪霍乱沙门氏菌。该方法利用葡萄糖氧化酶催化底物葡萄糖产生葡萄糖酸,导致溶液pH下降,进而引发溴甲酚紫（Bromocresol purple,BCP）溶液颜色由紫色变成亮黄色,随后通过记录BCP颜色变化（OD₄₃₀/OD₅₉₀）实现目标菌的定量检测。当菌浓度为2.54×10³~6.17×10⁵ CFU/mL,该方法定量检测猪霍乱沙门氏菌可用方程一表述：y₁=0.1051ln （x）+0.7024（R²=0.7513）;当菌浓度为6.17×10⁵~1.67×10⁷ CFU/mL,该方法定量检测猪霍乱沙门氏菌可用方程二表述：y₂=1.3216ln （x）-15.797（R²=0.9711）;当菌浓度大于1.67×10⁷ CFU/mL时,BCP溶液呈现明显亮黄色,OD₄₃₀/OD₅₉₀值趋于稳定,无法实现猪霍乱沙门氏菌定量检测。将六个不同浓度的猪霍乱沙门氏菌（2.5×10³~5.6×10⁶ CFU/mL）加标至牛奶中,检测结果显示回收率介于72.16%~103.58%,相对标准偏差介于7.54%~15.30%。总之,本研究所构建的比色ELISA方法适用于牛奶中不同浓度的猪霍乱沙门氏菌快速、灵敏定量检测。     

Effect of X-ray treatments on inoculated Escherichia coli O157: H7, Salmonella enterica, Shigella flexneri and Vibrio parahaemolyticus in ready-to-eat shrimp

This study was conducted to evaluate the inactivation effect of X-ray treatments on Escherichia coli O157: H7, Salmonella enteric (S. enterica), Shigella flexneri (S. flexneri) and Vibrio parahaemolyticus (V. parahaemolyticus) artificially inoculated in ready-to-eat (RTE) shrimp. A mixed culture of three strains of each tested pathogen was used to inoculate RTE shrimp. The shrimp samples were inoculated individually with selected pathogenic bacteria then aseptically placed in sterile plastic cups and air-dried at 22 °C for 30 min (to allow bacterial attachment) in the biosafety cabinet prior to X-ray treatments. The inoculated shrimp samples were then placed in sterilized bags and treated with 0.1, 0.2, 0.3, 0.5, 0.75, 1.0, 2.0, 3.0 and 4.0 kGy X-ray at ambient temperature (22 °C and 60% relative humidity). Surviving bacterial populations were evaluated using a non-selective medium (TSA) with the appropriate selective medium overlay for each bacterium; CT-SMAC agar for E. coli O157: H7, XLD for S. enterica and S. flexneri and TCBS for V. parahaemolyticus. More than a 6 log CFU reduction of E. coli O157: H7, S. enterica, S. flexneri and V. parahaemolyticus was achieved with 2.0, 4.0, 3.0 and 3.0 kGy X-ray, respectively. Furthermore, treatment with 0.75 kGy X-ray significantly reduced the initial microflora on RTE shrimp samples from 3.8 ± 0.2 log CFU g⁻¹ to less than detectable limit (<1.0 log CFU g⁻¹).     

基于适配体拉曼光谱技术快速检测肠炎沙门氏菌方法

目的:应用SELEX技术筛选高亲和力、高特异性适配体,利用该适配体结合拉曼光谱技术建立肠炎沙门氏菌快速检测方法。方法:采用全细菌指数富集的配体系统进化技术(whole-bacteria systematic evolution of ligands by exponential enrichment,whole-bacteria-SELEX)筛选肠炎沙门氏菌特异性核酸适配体,并采用酶联免疫吸附实验(enzyme-linked immuno sorbent assay,ELISA)与SERS技术对筛选出的适配体亲和力及特异性进行评价,建立肠炎沙门氏菌检测方法。结果:本研究通过对肠炎沙门氏菌进行十五轮SELEX筛选,并通过ELISA对其亲和力进行评价,筛选出Aptamer₄、Aptamer₁₀、Aptamer₁₂三条候选适配体,并通过SERS技术确认Aptamer₄为亲和力最佳适配体;将Aptamer₄与肠炎沙门氏菌、肺炎克雷伯氏菌等5种混合菌结合,结果表明,通过SERS技术可特异的检测出肠炎沙门氏菌,且该方法重复性较好,其最低检测限的细菌浓度为10² CFU/mL。且在猪肉样品的检测中,肠炎沙门氏菌的回收率为93.37%~100.18%。结论:应用SELEX方法成功筛选出与肠炎沙门氏菌高特异性、高亲和力适配体,并建立基于表面增强拉曼光谱技术快速检测肠炎沙门氏菌的方法,该方法特异性强、灵敏度高、成本低、快速简便,可应用于食品加工过程中肠炎沙门氏菌的快速检测。     

沙门菌、金黄色葡萄球菌、副溶血性弧菌的选择性共增菌培养基的研制

目的 研制一种可同时对沙门菌、金黄色葡萄球菌及副溶血性弧菌进行富集的共增菌培养基SSV,达到同时检测3种食品中常见的食源性致病菌的目的。方法 根据3株菌的营养需求,选择不同的培养基组分进行单因素实验,确定选择性共增菌培养基的配方,通过OD600 nm、受损菌复苏、多重聚合酶链式反应(PCR)、培养基选择性4个方面对SSV培养基进行验证。结果 确定增菌培养基的成分为：蛋白胨10.0 g、KH₂PO₄ 1.5 g、NaCl 15.0 g、LiCl 1.0 g,Na₂S₂O₃ 5.0 g、去氧胆酸钠0.05 g、甘露醇1.0 g、丙酮酸钠5.0 g,蒸馏水1 000 mL。SSV培养基能同时富集以上3株菌,37℃培养16 h后,3株菌的菌体浓度均达到107 CFU/mL及以上;SSV培养基对于受损的目标菌有良好的复苏效果,复苏后OD₆₀₀ nm较于复苏前增长了3倍;同时多重PCR、培养基选择性也得到很好的验证。结论共增菌培养基SSV可用于同时培养沙门菌、金黄色葡...     

Comparative analysis of acid resistance in Listeria monocytogenes and Salmonella enterica strains before and after exposure to poultry decontaminants. Role of the glutamate decarboxylase (GAD) system

Data on the ability of chemical poultry decontaminants to induce an acid stress response in pathogenic bacteria are lacking. This study was undertaken in order to compare the survival rates in acid broths of Listeria monocytogenes and Salmonella enterica strains, both exposed to and not exposed to decontaminants. The contribution of the glutamate decarboxylase (GAD) acid resistance system to the survival of bacteria in acid media was also examined. Four strains (L. monocytogenes serovar 1/2, L. monocytogenes serovar 4b, S. enterica serotype Typhymurium and S. enterica serotype Enteritidis) were tested before (control) and after exposure to trisodium phosphate, acidified sodium chlorite, citric acid, chlorine dioxide and peroxyacids (strains were repeatedly passed through media containing increasing concentrations of a compound). Stationary-phase cells (10⁸ cfu/ml) were inoculated into tryptic soy broth (TSB) acidified with citric acid (pH 2.7 and 5.0) with or without glutamate (10 mM) added, and incubated at 37 °C for 15 min. Survival percentages (calculated from viable colonies) varied from 2.47 ± 0.67% to 91.93 ± 5.83%. L. monocytogenes cells previously exposed to acid decontaminants (citric acid and peroxyacids) showed, when placed in acid TSB, a higher (P < 0.05) percentage of survival (average 38.80 ± 30.52%) than control and pre-exposed to non-acidic decontaminants strains (22.82 ± 23.80%). Similar (P > 0.05) survival percentages were observed in previously exposed to different decontaminants and control Salmonella strains. The GAD acid resistance system did not apparently play any role in the survival of L. monocytogenes or S. enterica at a low pH. This study demonstrates for the first time that prior exposure to acidic poultry decontaminants increases the percentage of survival of L. monocytogenes exposed to severe acid stress. These results have important implications for the meat industry when considering which decontaminant treatment to adopt.     

鼠伤寒沙门氏菌生物被膜的清除效果研究

目的:比较食品工业中常用的消毒剂、常用的防腐剂及几种天然成分对鼠伤寒沙门氏菌（Salmonella typhimurium）生物被膜的影响。方法:通过在培养过程中添加不同种类、不同浓度的清除剂,测定所选清除剂对生物被膜形成过程的影响;用所选的清除剂对成熟生物被膜进行浸泡处理,评估每种清除剂不同含量下的清除效果;利用噻唑蓝（Methylthiazolyldiphenyl-tetrazolium bromide,MTT）比色法测定经不同清除剂处理后生物被膜菌内细胞的代谢活性;采用稀释平板涂布法计算不同物质处理后生物被膜内细菌的存活数。结果:消毒剂乙醇（50%）、过氧化氢（1%）、苯扎氯铵（0.6%）、苯扎溴铵（0.6%）,天然成分香芹酚、肉桂醛和百里香精油（1.0 μL/mL）在较低浓度下即可有效抑制S. typhimurium CDC3和S. typhimurium ST34生物被膜的产生;消毒剂洗洁精、过氧化氢和天然成分香芹酚、肉桂醛和百里香精油在清除成熟生物被膜方面表现出了一定的优势,其中清除效率最理想的是过氧化氢,最大可达90%左右,洗洁精最大清除率可至80%,香芹酚、肉桂醛和百里香精油可达70%左右;消毒剂乙醇、苯扎氯铵和苯扎溴铵,以及天然成分香芹酚、肉桂醛和百里香精油可以明显降低生物被膜菌的细胞代谢活性及活菌存活数,可将生物被膜内活菌数降低2~3个lg值。结论:对于成熟生物被膜的清除,在初始的培养中添加清除剂或是天然成分可以更加完全有效地控制生物被膜的产生。     

Combined treatment of fumaric acid with aqueous chlorine dioxide or UV-C irradiation to inactivate Escherichia coli O157:H7, Salmonella enterica serovar Typhimurium, and Listeria monocytogenes inoculated on alfalfa and clover sprouts

Effect of fumaric acid, chlorine dioxide (ClO₂), and UV-C treatment was examined on the inactivation of microorganisms in alfalfa and clover sprouts. Clover sprouts were irradiated with UV-C light (1–10 kJ/m²), and the treatment decreased the population of total aerobic bacteria by 1.03–1.45 log CFU/g. Clover sprouts inoculated with pathogenic bacteria were treated with various concentration of fumaric acid, and 0.5 g/100 ml fumaric acid treatment was the most effective. In addition, the combined treatment of fumaric acid (0.5 g/100 ml)/UV-C (1 kJ/m²) reduced the populations of Escherichia coli O157:H7, Salmonella enterica serovar Typhimurium, and Listeria monocytogenes inoculated on clover sprouts by 3.02, 2.88, and 2.35 log CFU/g. Alfalfa sprouts were treated with ClO₂, fumaric acid, and the combination of fumaric acid/ClO₂. The combined treatment was the most effective, and it reduced the total aerobic bacteria by 3.18 log CFU/g as well as the initial populations of E. coli O157:H7, S. typhimurium, and L. monocytogenes inoculated on alfalfa sprouts by 4.06, 3.57, and 3.69 log CFU/g. These results suggest that the combined treatment of fumaric acid with UV-C or ClO₂ can be useful for improving the microbial safety of alfalfa and clover sprouts.     

CRISPR/Cas生物传感器检测食源性病原体的研究进展

食源性病原体引起的食源性疾病严重威胁着人类的健康,灵敏且快速检测食源性病原体成为预防疾病发生的重要手段。生物传感器具有灵敏度高,无需富集即可实时定量,易于现场检测等优点。CRISPR/Cas系统与生物传感器结合可提高生物传感器检测的灵敏度和准确性。本文介绍CRISPR/Cas系统的分类及作用机制,回顾基于CRISPR/Cas的各种生物传感器检测食源性病原体的方法,包括荧光传感器、电化学传感器和侧流层析试纸,分析应用不同Cas蛋白检测的优缺点,为食源性病原体的检测提供借鉴。     

Salmonella enteritidis: Virulence factors and invasive infection in poultry

Worldwide, Salmonella enteritidis is a clinically prevalent Salmonella serotype, which is associated with the consumption of foods containing eggs or poultry meat from systemically infected chickens. Several factors, including lipopolysaccharide structure, plasmid carriage and the expression of fimbriae, have been investigated in relation to their roles in pathogenicity. While each of these factors has been linked to virulence in different hosts, most have been discounted as playing a part in the systemic colonization of poultry. It is possible that recent acquisition of virulence factors, in combination with existing mechanisms, has contributed to the development of systemic infection with S. enteritidis and its consequent prevalence in poultry. The expression of certain fimbrial structures appears to be linked most strongly with systemic colonization in chickens, although the expression of other factors is necessary for infection.     

乙型副伤寒沙门氏菌在鲜切黄瓜中的生长行为及预测模型建立

为建立不同温度条件下鲜切黄瓜中乙型副伤寒沙门氏菌的生长预测模型,将新鲜黄瓜切丁,添加乙型副伤寒沙门氏菌,分别在10、15、20、25、30和35℃下的恒温条件下贮藏,以观察细菌的生长。使用USDA综合病原体建模程序(USDA-IPMP)拟合每个温度下每种细菌的生长曲线,以找出描述该细菌生长的最适初级生长模型,并拟合得到最大比生长速率。通过温度对初级模型中最大比生长速率的生长动力学拟合,分别建立Ratkowsky、Huang rate、Cardinal、Arrhenius-type二级生长模型,并进行数学评估和实测样品验证。结果表明,实验数据和生长曲线显示乙型副伤寒沙门氏菌的生长表现出三个阶段,包括延滞期,指数期和稳定期。乙型副伤寒沙门氏菌的延滞期时间随着孵育时间的增加而降低。相反,乙型副伤寒沙门氏菌的生长速率随着孵育温度而增加,由此表明风险随温度的升高而增加。使用Baranyi和Huang初级模型分析两种病原体的生长曲线,使用Ratkowsky、Huang平方根模型、Cardinal和Arrhenius模型描述温度对贮藏时间细菌生长的影响,同时应用实验数据和样品实测验证评估所建立的预测模型。从该研究中获得的结果和预测模型可用于预测鲜切黄瓜产品中乙型副伤寒沙门氏菌的生长。     

佳木斯市肠炎沙门菌的耐药特征及同源性分析

目的 了解佳木斯市优势血清型肠炎沙门菌的耐药状况及进化关系,为临床治疗提供科学依据。方法 对来自食品和腹泻患者的沙门菌进行血清学分型,采用微量肉汤稀释法对分离的肠炎沙门菌进行16种抗生素药敏试验,脉冲场凝胶电泳分析菌株之间的进化关系。结果 在108株沙门菌中,检出肠炎沙门菌55株（50.93%）;49株肠炎沙门菌有耐药性,耐药率为89.09%,其中33株菌（67.35%）为多重耐药,对抗生素萘啶酸和氨苄西林的耐药率较高,分别为81.82%和60.00%;55株肠炎沙门菌同源性为37%~100%。结论 佳木斯地区肠炎沙门菌多重耐药率高,菌株之间亲缘关系和耐药性存在密切关联性,来自食品和腹泻患者的肠炎沙门菌有交叉感染现象,依据本地区肠炎沙门菌耐药特征,制定有针对性诊疗方案和预防控制措施,对保障公共卫生健康发展具有重要意义。     

Growth of Salmonella enterica Serovar Enteritidis PT4 in media containing glucose results in enhanced RpoS-independent heat and acid tolerance but does not affect the ability to survive air-drying on surfaces

The heat and acid tolerance of isolates of Salmonella enterica serovar Enteritidis PT4 was markedly higher if cells were grown to stationary phase in either commercially produced Tryptone Soya (TSBG) or brain–heart infusion (BHI) broth containing glucose than when cells were grown in either nutrient or Tryptone Soya broth without glucose. Differences between the populations were found to be related to the production of acid in either TSBG or BHI which caused the culture media pH values to fall to pH 5·8–4·7 during overnight incubation. This led to habituation, which increased both heat- and acid-tolerance, although there was no effect on the abilities of cells to survive air-drying on surfaces. The acid tolerance mechanisms, induced by fermentative growth in complex media, responsible for survival at pH 2·8, were RpoS-independent. In addition, although growth rates as measured by cfu were essentially the same in all media, optical density measurements were very different, with those of cells in media containing glucose being much higher. This was found to be due to the formation of larger cells by the Salmonella in these media.     

The fate of Salmonella enteritidis PT4 in deliberately infected commercial mayonnaise

A total of 30 samples (24 different varieties) of commercial mayonnaise deliberately inoculated with Salmonella enteritidis PT4 have been studied. The fastest death rate of the test organism was obtained with a fat-free mayonnaise Ph 2·6. There was a trend indicating that the antimicrobial attributes of mayonnaise were enhanced when the pH became more acid, especially with acetic acid as the acidulant. The looseness of the correlation of death rates with pH in the range 4-5 may well reflect the contribution of the diverse range of ingredients other than acids to the antimicrobial properties of this commodity. One general statement is possible, in the majority of mayonnaises S. enteritidis was protected to a limited extent by storage at 4°C viz à viz storage at 20°C.     

Individual-based modelling of growth and migration of Salmonella enteritidis in hens' eggs

An individual-based model (IbM) was developed to describe the growth and migration of Salmonella enteritidis in hens' eggs. The Bacteria Simulator (BacSim) environment was used to implement the model; the bacteria are represented by spheres that grow by nutrient uptake and divide in two daughter cells upon exceeding a certain threshold volume. Motility of the Salmonella bacteria was described by a run and tumble mechanism. For the sake of simplicity, the bacteria were assumed to grow exponentially, an appropriate assumption for the initial phase of growth relevant for shelf-life predictions. Both albumen and yolk were assumed to be homogeneous. The impact of several model parameters (chemotaxis, growth rate, initial contamination numbers and bacterial swimming speed) was assessed by a sensitivity analysis. The results show that chemotaxis towards the yolk would have a strong effect on the time needed to reach the vitelline membrane, an aspect that future research should focus on. The contamination position had less impact on the time to reach the vitelline membrane. The simulation results illustrate the need for more detailed knowledge on the subject of bacterial migration in hens' eggs. Our model can easily incorporate this knowledge when it becomes available.     

生物传感器在食源性金黄色葡萄球菌快速检测中的应用

金黄色葡萄球菌(Staphylococcus aureus)是引起食源性疾病爆发的重要致病菌之一,传统的金黄色葡萄球菌检测方法包括培养法、生化鉴定法、核酸分析法、免疫分析法等,其可靠性高,但操作相对繁琐、费时,且灵敏度较低.生物传感器是一种由生物学、光学、化学等多学科交叉渗透发展形成的新型微量分析技术,具有灵敏度高、分...     

电化学生物传感器在黄曲霉毒素检测中的研究进展

黄曲霉毒素是黄曲霉和寄生曲霉的次级代谢产物,具有剧毒性和严重的致癌性、致畸性和致突变性,摄入受黄曲霉毒素污染的农作物和食品会对人类和动物的身体健康造成极其严重的危害。基于十年来国内外的研究成果,从电化学传感机理方面概述了电化学生物传感器在黄曲霉毒素检测中的发展现状和应用前景,介绍了各类电化学生物传感器的工作原理、制备方法和检测性能。电化学生物传感器相比于传统的黄曲霉毒素检测方法,具有检测时间短、操作简单、成本低、便于微型化等优势。金纳米颗粒、碳纳米管等纳米材料的使用将进一步提高电化学生物传感器的灵敏度以及抗原或抗体的固定效率。超微电极技术、传感器阵列技术以及微流控技术的应用也将进一步提高电化学生物传感器在毒素检测中的性能。     

三文鱼来源副溶血性弧菌的血清分型、毒力基因及耐药性研究

目的 对2019年广州市售三文鱼中副溶血性弧菌的血清型、毒力基因分布及耐药特征进行研究,为防控因食用三文鱼引起副溶血性弧菌食源性疾病提供数据支持。方法 根据GB 4789.7—2013方法对90份广州市售三文鱼样品进行副溶血性弧菌分离、定量及血清分型,分离株的鉴定及耐药性分析采用全自动微生物鉴定仪分析,通过PCR扩增对毒力基因进行检测。结果 90份三文鱼样品中,有16份检出副溶血性弧菌,总检出率为17.78%,阳性样品污染量范围为3.6~93 MPN/g,其中批发市场的检出率（27.50%）最高。MPN计数阳性管分离得到的72株副溶血性弧菌经血清凝集共分出11个O群血清型,分型率为80.56%,其中O11、O9和O6群为主要血清型,占51.39%,其他血清型分布较分散。72株副溶血性弧菌均含有tlh基因,且均未检测到trh和tdh基因。药敏分析发现,全部分离株对碳青霉烯类、单酰胺环类、氨基糖苷类、喹诺酮类和硝基呋喃类等5类18种抗生素敏感。分离株对氨苄西林高度耐药,耐药率达97.22%,少数分离株对哌拉西林（1.39%）和复方新诺明（5.56%）耐药。6.94%的分离株同时对2种抗生素耐药,未出现多重耐药。结论 广州市售三文鱼受到副溶血性弧菌污染程度较高,分离株的血清型呈多样化,主要为非致病株,但具有一定程度的耐药,存在危害健康的潜在风险。     

一起肠炎沙门菌引起的校园食源性疾病暴发事件调查溯源分析

目的 调查一起肠炎沙门菌引起的学校食源性疾病暴发事件,并对危险因素开展分析溯源,为预防类似事件的发生提供科学依据。方法 运用描述性流行病学方法分析事件的流行病学特征;运用病例对照研究调查可疑食物;通过环境卫生学方法调查追溯食品污染的过程;对病例、食物、环境样品进行病原菌分离、血清分型和脉冲场凝胶电泳（PFGE）检测。结果 本起暴发事件共报告病例71例,流行曲线符合持续同源暴发模式的特点。病例对照、环境卫生调查和病因溯源研究提示学校超市售卖的三明治是引起本次事件的危险食品（OR=302.09,95%CI=75.18~1 213.97）。采集的病例、食品、环境样品中有8份（12.70%,8/63）检出了肠炎沙门菌,分离菌株的PFGE图谱条带完全一致,证实了学校超市出售的三明治使用的沙拉酱在制作过程中被肠炎沙门菌污染,是导致这次食源性疾病暴发的原因。结论 本次事件是由肠炎沙门菌污染食品引起的食源性疾病暴发,可疑食品为被自制沙拉酱污染的三明治。建议禁止各类未经充分热处理的含生鲜鸡蛋成分的食品进入学校,相关行政部门应加强对该类食品的卫生监督。