肠炎沙门氏菌rpoH 基因缺陷株的构建及其热激响应特性

构建肠炎沙门氏菌Salmonella enteritidis IFO3313 株的rpoH 基因缺陷株IFO3313- ΔrpoH,比较不同温度下缺陷株与野生株的热激响应特性。利用λ-Red 重组系统对Salmonella enteritidis IFO3313 的rpoH 基因进行缺失突变,并使用PCR方法对其进行验证,在此基础上使用不同培养基对缺陷株与野生株进行不同温度下的热激应答结果和亚致死热损伤修复能力的考察：野生株比缺陷株有更强的热激耐受能力,在DHL 平板上对亚致死热损伤的肠炎沙门氏菌的分离检测可能具有假阴性,野生株比缺陷株在TSYA 平板上有更强的修复能力。利用λ-Red 重组系统敲除了肠炎沙门氏菌rpoH 基因,有助于了解肠炎沙门氏菌的热激亚致死及其修复机制,对亚致死状态食源性病原微生物的检测做出初步研究。     

沙门氏菌热裂解气相色谱-质谱法研究

文中应用热裂解气相色谱-质谱技术,对沙门氏菌属中的4种菌株(甲型副伤寒、乙型副伤寒、鼠伤寒和G1M1.345肠炎沙门氏菌肠炎亚种)进行了研究,探讨了热裂解气相色谱-质谱的条件,得到了清晰的总离子流色谱图,确定了部分裂解产物的分子结构。结果表明:这4种沙门氏菌全细胞在裂解温度650℃,裂解时间12 s,离子源温度230℃的条件下均可得到清晰的总离子流色谱图,且这4种沙门氏菌的色谱图相似度极高;另通过质谱分析和比对,确定了这4种沙门氏菌裂解产物包括苯酚、吲哚和1-十三烯的分子结构式。总体来说,该方法可作为沙门氏菌属的快速检测,但不能用于属内菌株类别的鉴定。     

屠宰场生鸡肉肠炎沙门氏菌的污染途径

目的：研究屠宰场生鸡肉肠炎沙门氏菌的主要污染途径。方法：以屠宰场生鸡肉样品为研究对象,在利用多聚酶链反应(PCR)检测肠炎沙门氏菌污染的基础上,通过流行病学的方法研究生鸡肉污染与肉鸡感染的关系。结果：屠宰场生鸡肉样品肠炎沙门氏菌污染阳性率为9.03%(26/288),样品污染与样品来源屠宰场间无统计学联系,但与养殖场的肉鸡感染肠炎沙门氏菌有联系;养殖场肉鸡感染肠炎沙门氏菌与生鸡肉污染有正的统计学联系(相对危险性=18.5);肉鸡实验性感染肠炎沙门氏菌后的带菌时间可达42d,排菌达56d 以上。结论：屠宰场生鸡肉肠炎沙门氏菌的污染途径主要为内源性污染。     

食品中热损伤沙门氏菌选择性增菌及实时荧光聚合酶链式反应检测

为了能够高效检测食品中的亚致死损伤沙门氏菌,首先采用热激胁迫的方法得到亚致死损伤沙门氏菌细胞,进而基于选择性增菌培养液SEL建立一种实时荧光聚合酶链式反应检测技术。结果表明：在SEL中经过20h增菌培养后,无论是否经过修复培养,1～2CFU/5mL SEL的亚致死损伤细胞都能得到完全修复并增菌至109CFU/mL水平;依此建立的实时荧光聚合酶链式反应检测技术的纯菌扩增效率为95.41%,检测限为4CFU/反应体系,在人工污染碎食品样品中的检测限为3CFU/10g碎牛肉,而且与传统培养检测方法的结果相吻合。本方法在24h内即可完成食品中热损伤沙门氏菌的修复、选择性增菌以及实时荧光聚合酶链式反应检测,可应用于食品中沙门氏菌污染状况调查及高效检测。     

3种血清型沙门氏菌多重PCR检测方法的建立

目的建立多重聚合酶链式反应(multiplex polymerase chain reaction,mPCR)法检测3种常见血清型沙门氏菌的分析方法。方法以肠炎沙门氏菌Hat基因、鼠伤寒沙门氏菌Stm-4495基因、乙型副伤寒沙门氏菌sdfI基因设计合成特异性引物,提取沙门氏菌基因组DNA为扩增模板,验证引物特异性,优化多重PCR退火温度及引物浓度,检测方法特异性及检出限,并利用该方法对人工污染冷鲜鸡肉进行检测。结果 3对引物特异性强且无交叉影响;25μL多重反应体系中,引物STM、HAT、SDF最佳终浓度分别为:0.5、0.5、0.4μmol/L,最佳退火温度为60℃;11株阴性对照菌无目标条带检出,方法特异性良好,以3种血清型沙门氏菌纯培养物DNA混合物为模板,检出限低至DNA质量浓度1 pg/μL;人工污染鸡肉经过12 h增菌,能同时检测出3种血清型沙门氏菌的检测限为:肠炎沙门氏菌(4.0±1.0) CFU/g、鼠伤寒沙门氏菌(8.0±0.9) CFU/g、乙型副伤寒沙门氏菌(8.0±0.6) CFU/g。结论本方法特异性强、检测限低,对食品中3种常见血清型沙门氏菌快速检测具有重要意义。     

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。     

生菜表面肠炎沙门氏菌(ATCC13076)的吸附及其清除

为了控制生鲜蔬菜上沙门氏菌的污染,本研究以肠炎沙门氏菌(ATCC13076)为研究对象,探索了其在生菜表面吸附的影响因素,同时比对了含氯消毒剂和裂解噬菌体对生菜上粘附沙门氏菌的清除效果。结果表明:随着温度的升高(4～32℃)和作用时间的延长(5～60 min),肠炎沙门氏菌在生菜表面的吸附量呈增加趋势(3.59～5.20 log cfu/cm~2)。用10 mg/mL次氯酸钠处理受污染的生菜,在作用2 h时达到最大清除量(3.83 log cfu/cm~2),随着作用时间的延长(0～48 h)清除量呈不断下降的趋势;而噬菌体随着感染复数MOI值的增加(0.1、1、10)与作用时间的延长(0～48 h),对肠炎沙门氏菌的清除量逐渐增加,在4℃和24℃时,对肠炎沙门氏菌的最高清除量分别为1.75 log cfu/cm~2和1.80 log cfu/cm~2。因此,肠炎沙门氏菌在生菜上的吸附量受到温度、作用时间和pH的影响,与含氯消毒剂相比,噬菌体可以安全有效的清除生菜上沙门氏菌。本研究可以为食源性病原微生物在生鲜蔬菜中的清除提供理论支撑与技术指导。     

质粒介导的氨苄西林耐药肠炎沙门氏菌生长特性及抗性研究

肠炎沙门氏菌是一种重要食源性致病菌，其耐药性成为全球关注的问题。耐药质粒的广泛传播提高了肠炎沙门氏菌的耐药性，耐药质粒的获得可能会改变细菌的生长特性及对环境压力的抗性，这给耐药食源性致病菌的控制及风险评估带来了新的挑战。为探究质粒介导的氨苄西林耐药性对肠炎沙门氏菌生长以及抗性变化的影响，该研究将含有氨苄西林抗性基因的pKD46质粒导入到抗生素敏感的肠炎沙门氏菌中，获得氨苄西林耐药菌株。分别对转化后的耐药沙门氏菌的生长特性、酸与热抗性进行检测。结果发现耐药质粒转化后的肠炎沙门氏菌菌株对其他种类抗生素的抗性基本不变；生长的最大比生长速率(μ_max)不变，生长延滞期(λ)延长；在57.5与60℃条件下的热抗性显著降低；对pH 3.0的酸抗性未发生显著改变。研究结果发现质粒介导的氨苄西林耐药性可降低肠炎沙门氏菌的生长及热抗性，该结果可为耐药食源性致病菌的控制及风险评估提供参考。     

免疫磁纳米粒子对肠炎沙门氏菌的富集分离

采用化学共沉淀法制备了羧基化磁纳米粒子,分别对磁纳米粒子-沙门氏菌多克隆抗体复合物（免疫磁纳米粒子）的偶合条件和免疫磁纳米粒子富集分离肠炎沙门氏菌的条件进行了优化,为肠炎沙门氏菌的富集分离和检测提供一种更为快捷、高效的方法。结果表明,当51.7 μg/mL羧基化磁纳米粒子与碳二亚胺/N-羟基丁二酰亚胺（0.4 mol/L/0.1 mol/L）、1.0 mg/mL的多克隆抗体的体积比为1∶2∶2时,37 ℃水浴加热40 min,两者的偶合效果最佳。应用上述优化条件制备的免疫磁纳米粒子吸附104 CFU/mL肠炎沙门氏菌,当两者的体积比为4∶5,孵育时间为40 min时,免疫磁纳米粒子对肠炎沙门氏菌的吸附效率可达到94.36%。     

基于核酸适配体的纳米金比色法快速检测肠炎沙门氏菌

该文以肠炎沙门氏菌为研究对象,开发基于核酸适配体的纳米金可视化检测方法。通过优化体系内适配体浓度,研究纳米金-适配体体系的肠炎沙门氏菌检测限、特异性及适用温度;同时以人工污染样品为例,评价纳米金-适配体的加标回收率。结果显示：该方法可以特异性检测肠炎沙门氏菌,对其他食源性致病菌无特异反应。通过条件优化,在适配体浓度200 nmol/L 下,肠炎沙门氏菌的最低检测限为9.3×101 CFU/mL,其线性范围为103～107 CFU/mL,线性方程为y=0.187 8x-0.146（R2=0.991 3）。检测人工污染样品的加标回收率为93.68%～117.89%。利用核酸适配体纳米金比色法进行肠炎沙门氏菌的检测操作简便、结果可视;通过调整核酸适配体可进行其他致病菌的检测,具有良好的推广意义。     

Thermal injury and recovery of Salmonella enterica serovar enteritidis in ground chicken with temperature, pH, and sodium chloride as controlling factors

Cells of Salmonella enterica serovar Enteritidis were grown at 25 and 35 degrees C, heat injured (55, 60, and 62.5 degrees C), and recovered in tryptic soy broth (TSB) at various NaCl concentrations (2.0 and 3.5%) and pH levels (5.5 and 6.5). To assess the interactions of growth temperature, heating temperature, NaCl concentration and pH on the thermal injury and recovery of Salmonella Enteritidis in ground chicken, a randomized design with each experimental combination was used. When a logistic equation for nonlinear survival curves was used, D-values of cells of Salmonella Enteritidis grown at 25 degrees C were 7.60, 5.73, and 4.81 min at 55, 60, and 62.5 degrees C, respectively. For cells grown at 35 degrees C, the D-values were 12.38, 7.45, and 5.70 min at 55, 60, and 62.5 degrees C. The influence of tryptic soy agar and double modified lysine agar (DMLIA) on the recovery of heat-injured cells was determined. Recovery was significantly reduced on DMLIA at increased pH levels and NaCl concentrations. Higher numbers of cells were recovered in TSB with 2.0% NaCl than in TSB with 3.5% NaCl. It was observed that the rate of recovery of heat-injured cells was similar at each pH. Therefore, a pH range of 5.5 to 6.5 does not have a major inhibitory effect on the recovery of Salmonella Enteritidis.     

Simultaneous Recovery of Four Heat-injured Foodborne Pathogens from a Four-compartment Thin Agar Layer Plate

ABSTRACT: We have developed and tested a 1-step resuscitation thin agar layer (TAL) system (nonselective agar overlaid on selective agar) to effectively recover heat-, acid-, cold-, and salt-injured foodborne pathogens in liquid foods. Each TAL unit was designed for the recovery of one injured population. To improve operation efficiency we have developed a single 4-compartment Petri dish to simultaneously recover heat-injured Escherichia coli O157:H7, Listeria monocytogenes, Salmonella Typhimurium , and Yersinia enterocolitica. No significant difference occurred between the single recovery unit and the 4-compartment unit for enumeration of 4 heat-injured pathogens (P > 0.05). This 4-compartment TAL system is at least 4 times more efficient than the single TAL system.     

Application of thin agar layer method for recovery of injured Salmonella typhimurium

Xylose lysine decarboxylase (XLD) medium, a selective plating medium, can inhibit heat-injured Salmonella typhimurium from growing, whereas tryptic soy agar (TSA), a nonselective medium, does not. To facilitate recovery of heat-injured S. typhimurium cells while providing selectivity of isolation of S. typhimurium from other bacteria in the sample, a thin agar layer (TAL) procedure was developed by overlaying 14 ml of nonselective medium (TSA) onto prepoured and solidified XLD medium in a 8.5 cm diameter Petri dish. During the first few hours of incubating the plate, the injured S. typhimurium repaired and started to grow in the TSA. During the resuscitation of injured cells, the selective agents from XLD were diffused to the TSA top layer part. Once the selective agents diffused to the top part of the TAL, the resuscitated S. typhimurium started to produce a typical reaction (black color) and other microorganisms were inhibited by the selective agents. The recovery rate for heat-injured (55 degrees C for 15 min) S. typhimurium with the TAL method was compared with TSA, XLD, and the traditional overlay method (OV; pouring selective agar on top of resuscitated cells on TSA agar 3-4 h after incubation). No significant difference occurred among TSA, OV, and TAL (P > 0.05) for enumeration of heat-injured S. typhimurium, but they recovered significantly higher numbers than from XLD agar (P < 0.05).     

基于纳米探针检测食源性肠炎沙门氏菌技术初探

目的建立纳米探针快速、准确检测食源性肠炎沙门氏菌的方法。方法利用二氧化硅磁纳米材料与肠炎沙门氏菌抗体制备纳米探针,将制备后的探针与经过荧光染色的肠炎沙门氏菌结合,并通过荧光显微镜和流式细胞仪对探针捕获的细菌进行检测,并比较2种方法的优劣。结果本方法设计合成了肠炎沙门氏菌纳米探针,通过荧光显微镜可以观察到复合纳米探针结合的5′106 CFU/mL和5′107 CFU/mL浓度的肠炎沙门氏菌,在放大400倍的显微镜视野中成倍递增,该探针结合流式细胞术可以检测到5′105 CFU/mL浓度的肠炎沙门氏菌。结论纳米探针技术结合荧光显微镜、流式细胞术可以更加快速、准确、灵敏地用于食源性肠炎沙门氏菌的定性检测,并有望通过进一步实验实现食源性致病菌的定量检测。     

肠炎沙门氏菌的LAMP检测方法的建立

建立肠炎沙门氏菌(Salmonella enteritidis)的环介导等温扩增(Loop-mediated isothermal amplification,LAMP)方法,实现对肠炎沙门氏菌的快速检测.通过针对肠炎沙门氏菌血清型特异性基因lygD设计LAMP内外引物对,优化LAMP扩增反应条件,采用包括肠炎沙门氏菌在内的10种不同菌株进行LAMP引物特异性检测;通过系列梯度稀释肠炎沙门氏菌菌液进行LAMP扩增,计算检出限;并对鲜鸡蛋模拟样本进行LAMP检测.结果表明LAMP法可快速特异地检测出肠炎沙门氏菌;细菌培养液检出限为2.33×101 cfu/mL,鲜鸡蛋模拟样品为2.67×l01cfu/mL.该方法反应灵敏度高,可用于食品中肠炎沙门氏菌的快速检测.     

Efficacy of disinfection of shell eggs externally contaminated with Salmonella enteritidis. Implications for egg testing.

Experimental contamination of the surface of shell eggs by dipping in a culture of Salmonella enteritidis resulted in the presence of Salmonella enteritidis in/on the shells as well as shell membranes but not in the egg content. Disinfection with Lugol's solution, chlorhexidine, ethanol, quarternary ammonium solutions or flaming after dipping in ethanol failed to achieve complete decontamination of the shell and membranes with resulting false positives when eggs were broken for culturing of the content. Dipping eggs for three seconds in boiling water resulted in complete destruction of Salmonella enteritidis in shells and membranes but sometimes caused the eggs to crack. A method of aseptically opening eggs without risk of contaminating the content from the shell or membrane was developed. Salmonella enteritidis deposited in/on the shell and membranes did not multiply during storage of the eggs at 20 degrees C for four weeks, the counts seemed to decrease. No Salmonella enteritidis was detected in the contents of any contaminated eggs.     

腹泻患儿粪便中沙门菌的血清型、药敏分析及脉冲场凝胶电泳分子分型研究

目的 研究2019年北京市朝阳区0-10岁腹泻患儿粪便中分离出的沙门菌的血清型、PFGE分子分型研究及耐药特点。方法 对分离自腹泻患儿病例的47株沙门菌进行血清分型,采用微量肉汤稀释法进行27种抗生素的药敏实验;采用PFGE脉冲场凝胶电泳进行指纹图谱分型研究。结果 47株沙门分为9种血清型,优势血清型两种,分别为肠炎沙门菌23株占48.94%,鼠伤寒沙门菌14株占29.79%。47株沙门菌对磺胺异恶唑耐药率(57.45%)最高,其次为氨苄西林(48.94%)和链霉素(48.94%)。23株肠炎沙门菌可分成8个PFGE指纹图谱,15株鼠伤寒沙门菌可分成14个PFGE指纹图谱。结论 北京市朝阳区0-10岁儿童食源性沙门菌血清主要为肠炎沙门菌和鼠伤寒沙门菌,各血清型的耐药性有所不同,PFGE指纹图谱呈多样性。