产气荚膜梭菌肠毒素基因地高辛探针制备与检测方法建立的初步研究

产气荚膜梭菌（ｃｌｏｓｔｒｉｄｉｕｍ ｐｅｒｆｒｉｎｇｅｎｓ,ＣＰ）是人类和家畜重要的厌氧性病原菌,产生肠毒素的ＣＰ是人类食物中毒病原菌。本研究用地高辛标记肠毒素基因制备探针,并建立了原位杂交检测方法,直接检测肠毒素基因（ｃｐｅ）。这是继ＰＣＲ检测方法后又一项特异性基因诊断技术。由于利用了光学显微镜观察结果,形态学上比较直观,增加了实验的准确性。与ＰＣＲ、乳胶凝集相比,原位杂交检测方法具有较高的敏     

肉类食品中产气荚膜梭菌及其控制研究进展

产气荚膜梭菌广泛存在于自然界,且可以形成芽孢,是导致肉类食物中毒和气性坏疽等的主要病原菌。本文综述了肉类食品中产气荚膜梭菌的污染情况、生长影响因素以及控制其生长的方法;指明了利用食用安全的天然物质,研发高效抑制产气荚膜梭菌生长的技术与方法,对保障肉制品安全和促进肉类工业健康发展的现实意义,旨在为人们选择适合肉类食品中抑制产气荚膜梭菌生长的途径提供参考。     

郑州市熟肉制品中产气荚膜梭菌污染状况调查

目的了解郑州市熟肉制品中产气荚膜梭菌(Clostridium perfringens)的污染状况,为熟肉制品储存过程中产气荚膜梭菌的安全控制提供技术支撑。方法按照GB 4789.13—2012《食品安全国家标准食品微生物学检验产气荚膜梭菌检验》中的方法分别对11种不同样品进行检测分析,结合VITEK鉴定法和分子生物学鉴定法进一步验证。结果 259份样品中,产气荚膜梭菌检出份数为38份,总检出率14.7%,不同样品的检出率范围为0.0%~33.3%,其中盐焗鸡样品检出率最高为33.3%(7/21),卤半片鸭和卤鸡肝样品未检出。烧鸡、烤鸡和卤鸡腿样品的产气荚膜梭菌菌落总数均超过5.0log10CFU/g,其他样品的菌落总数均在4log_(10)~5log_(10) CFU/g之间。结论郑州市熟肉制品中的产气荚膜梭菌污染现象较严重,烧鸡、烤鸡和卤鸡腿的菌落总数较高,有引起食物中毒的可能。     

产气荚膜梭菌质控样品的研制

目的 制备产气荚膜梭菌质控菌株质控样品.方法 将产气荚膜梭菌菌悬液于36℃厌氧培养至浓度为107 CFU/mL,以10％(m/m,下同)脱脂奶粉、5％海藻糖、5％蔗糖、1％谷氨酸钠、0.5％抗坏血酸钠为菌体保护剂,使用冷冻干燥法制备冻干菌剂.依据CNAS-CL03-A001:2019《能力验证提供者认可准则在微生物领域...     

荧光MIRA法快速检测食品中产气荚膜梭菌

目的 基于荧光多酶恒温快速扩增（MIRA）技术,建立一种快速准确检测食品中产气荚膜梭菌的方法。方法 利用产气荚膜梭菌共有α毒素编码基因plc保守序列设计引物探针,并建立和优化荧光MIRA方法;提取17种非目标菌基因组DNA进行方法特异性验证;梯度稀释目标菌基因组DNA和菌液用于方法灵敏度评价;选取4种食品进行基质干扰试验,评估方法的稳定性;检测实际样品并与国家标准方法进行比较,以验证该方法的实用性。结果 该方法特异性强,检测多种非目标致病菌均呈阴性反应;灵敏度高,以基因组DNA为模板和菌液为模板时最低检测限分别为1.05×101 fg/μL和7.5×100 CFU/mL;该方法不受基质影响,抗干扰能力强;耗时短,约20 min即可完成检测。结论 本研究中的荧光MIRA方法快速准确、灵敏度高、特异性强,可用于食品中产气荚膜梭菌的快速检测。     

环介导等温扩增技术快速检测产气荚膜梭菌的研究

目的:建立环介导等温扩增技术(LAMP)快速检测产气荚膜梭菌的方法。方法:以产气荚膜梭菌(ATCC13124)的α毒素全基因序列为保守序列,设计内、外、环引物,肉眼观察白色沉淀并判断检测结果。结果:LAMP检测产气荚膜梭菌的灵敏度为2.92×102CFU/mL,人工污染产气荚膜梭菌的全脂乳的检测限为15.7CFU/mL,耗时仅1h。对照PCR检测产气荚膜梭菌的灵敏度为2.92×105CFU/mL,人工污染产气荚膜梭菌的全脂乳的检出限1.57×105CFU/mL。采用同样的方法提取DNA,耗时3h。结论:建立LAMP快速、特异检测产气荚膜梭菌的方法,该方法灵敏度是PCR的1000倍,为食品中产气荚膜梭菌的检测构建了一个技术平台。     

聚赖氨酸对产气荚膜梭菌的抑菌研究

产气荚膜梭菌(Clostridiumperfringens)是真空包装熟肉制品中最常见的一种厌氧污染菌。研究表明,ε-聚赖氨酸对其具有很强的抑制作用,在最适抑菌pH下(pH6.5),ε-聚赖氨酸对产气荚膜梭菌的最小抑菌浓度(MIC)为0.025%;高价金属阳离子对ε-聚赖氨酸的抑菌活性有明显的抑制作用,而EDTA则可以络合高价金属离子,增强其抑菌作用。将0.01%的EDTA和0.01%的ε-聚赖氨酸复合使用时可完全抑制产气荚膜梭菌的生长繁殖。     

聚赖氨酸对产气荚膜梭菌的抑菌研究

产气荚膜梭菌（Clostridiumperfringens）是真空包装熟肉制品中最常见的一种厌氧污染菌。研究表明,ε-聚赖氨酸对其具有很强的抑制作用,在最适抑菌pH下（pH6.5）,ε-聚赖氨酸对产气荚膜梭菌的最小抑菌浓度（MIC）为0.025%;高价金属阳离子对ε-聚赖氨酸的抑菌活性有明显的抑制作用,而EDTA则可以络合高价金属离子,增强其抑菌作用。将0.01%的EDTA和0.01%的ε-聚赖氨酸复合使用时可完全抑制产气荚膜梭菌的生长繁殖。      

产气荚膜梭菌在食品中的危害及其控制研究进展

产气荚膜梭菌(Clostridium perfringens)是一种革兰氏阳性厌氧芽孢致病菌,广泛分布于环境、动物以及人类的胃肠道中,因能分解肌肉和结缔组织中的糖类并产出大量气体以及形成荚膜而得名.其对生长环境要求较低,是自然界中常见的致病菌.其芽孢是在环境胁迫(低温、干燥或营养缺乏等)下形成的一种微生物休眠体,具有极...     

一起疑似产气荚膜梭菌食物中毒事件的病原学分析

目的 分析一起疑似产气荚膜梭菌食物中毒事件的病原学。方法 利用荧光定量PCR方法对一起食物中毒患者的粪便进行产气荚膜梭菌基因的初筛, 根据荧光定量PCR初筛的提示结果进行细菌的分离培养, 对分离到的菌株进行质谱和生化鉴定、毒力基因检测和PFGE分析。结果 从5份食物中毒患者粪便中分离到5株携带肠毒素基因cpe的A型产气荚膜梭菌, 并且这5株菌的PFGE带型完全一致。结论 此次食物中毒致病因子为携带肠毒素基因cpe的A型产气荚膜梭菌, PFGE结果提示5株菌可能有同一来源。     

异硫氰酸苄酯对产气荚膜梭菌抑菌作用的研究

本文旨在研究异硫氰酸烯丙酯（Allyl isothiocyanate,AITC）对产气荚膜梭菌（Clostridium perfringens,C. perfringens）的抑菌效果及作用机制。首先,通过测定最小抑菌浓度（Minimum inhibition concentration,MIC）、绘制生长曲线评估AITC对C. perfringens的抑菌效果,并采用扫描电镜观察细胞形态、碘化丙啶染色实验测定细胞膜完整性,评估AITC对C. perfringens细胞膜的影响;进一步通过SDS-PAGE图谱分析和ATP酶活力测定研究AITC对C. perfringens细胞代谢的影响;最后,研究了AITC对熟猪肉糜中C. perfringens的抑制效果。结果表明,AITC可有效抑制C. perfringens的生长,其MIC为0.1 μL/mL;AITC引起C. perfringens细胞膜破裂、凹陷等形变,导致细胞膜完整性丧失,且膜损伤的程度随AITC浓度的升高而增强;AITC显著（P<0.05）降低了C. perfringens的蛋白质含量和ATP酶活性,进而影响细胞的正常代谢;此外,添加0.1%~0.4%的AITC能够显著（P<0.05）抑制熟猪肉糜中C. perfringens的生长。综上,AITC可以通过破坏C. perfringens的细胞膜和干扰蛋白质代谢达到抑菌效果,本研究为天然抑菌剂在肉类行业中的应用提供了一定的理论基础。

     

Nachweis und Charakterisierung von Clostridium perfringens mittels real-time-PCR

Clostridium perfringens is a Gram-positive, anaerobic bacterium. The strains are classified into five types (type A to E), depending on the ability to produce alpha-, beta-, epsiloon and iota-toxin, but food-poisonings are mostly caused by C. perfringens type A isolates. Ingestion of contaminated food is followed by gastrointestinal disease, when enzyme-resistant C. perfringens enterotoxins (CpE) are set free during sporulation. In most cases the bacterium has to grow up to more than 10⁶ cfu/g food to cause a gastrointestinal disease (BVET, 2005; BfR, 2005). Cultural methods, normally used for the detection of C. perfringens, are not able to differentiate isolates into the five different toxin types. This is the reason, why the detection of C. perfringens in food under the present legal regulations can only used as an indication for inadequate production hygiene or a toxin-infection with contaminated food as source. For the detection of the toxin genes to differentiate C. perfringens in five different toxin types, three duplex real-time-PCR assays for the routine diagnostic were developed and validated. The assays can be used for quick detection and easy classification of C. perfringens isolates and as a rapid screening-system in suspected cases of C. perfringens food-poisonings.     

产气荚膜梭菌磷脂酶C的重组表达及其脱胶应用

将产气荚膜梭菌(Clostridium perfringens)磷脂酶C(Cp-PLC)基因在大肠杆菌BL21(DE3)中进行了重组表达,并对重组Cp-PLC表达条件进行了优化。结果表明:当菌体生长至OD600为1.0时,添加8 g/L乳糖在30℃下诱导32 h,得到的重组Cp-PLC的酶活最大,为398.35 U/mL;重组Cp-PLC用于菜籽毛油脱胶的最佳条件为温度57℃、pH 5.0、加酶量600 U/kg、反应时间1.5 h;在最佳条件下,菜籽毛油的磷含量可降至5.66 mg/kg,能够满足物理精炼的要求。     

Description of growth of Clostridium perfringens in cooked beef with multiple linear models

The traditional linear model used in food microbiology employs three linear segments to describe the process of food spoilage and categorize a growth curve into three phases — lag, exponential, and stationary. The linear model is accurate only within certain portions of each phase of a growth process, and can underestimate or overestimate the transitional phases. While sigmoid functions (such as the Gompertz and logistic equations) can be used to fit the experimental growth data more accurately, they fail to indicate the physiological state of bacterial growth. The objective of this paper was to develop a new methodology to describe and categorize accurately the bacterial growth as a process using Clostridium perfringens as a test organism. This methodology utilized five linear segments represented by five linear models to categorize a bacterial growth process into lag, first transitional, exponential, second transitional, and stationary phases. Growth curves described in this paper using multiple linear models were more accurate than the traditional three-segment linear models, and were statistically equivalent to the Gompertz models. With the growth rates of transitional phases set to 1/3 of the exponential phase, the durations of the lag, first transitional, exponential, and second transitional phases in a growth curve described by the new method were correlated linearly. Since this linear relationship was independent of temperature, a complete five-segment growth curve could be generated from the maximum growth rate and a known duration of the first four growth phases. Moreover, the lag phase duration defined by the new method was a linear function of the traditional lag phase duration calculated from the Gompertz equation. With this relationship, the two traditional parameters (lag phase and maximum growth rate) used in a three-segment linear model can be used to generate a more accurate five-segment linear growth curve without involving complicated mathematical calculations.     

一起疑似产气荚膜梭菌腹泻暴发事件实验室检测分析

目的 分析一起疑似由产气荚膜梭菌导致腹泻暴发事件的实验室检测结果,为产气荚膜梭菌食物中毒实验室检测策略的改进奠定基础。方法 采集暴发事件中4例病例肛拭子样本,应用荧光PCR方法检测肛拭子及其增菌液中cpa基因与cpe基因。对肛拭子样本进行产气荚膜梭菌分离培养,对部分分离单菌落进行产气荚膜梭菌毒力基因检测和脉冲场凝胶电泳（PFGE）分子分型。结果 4例病例样本中均检测到cpa基因和cpe基因。从病例1样本中挑取并鉴定为产气荚膜梭菌的18个单菌落中获得1个cpe+菌落,构成比为5.56%（1/18）;从病例2样本中挑取并鉴定为产气荚膜梭菌的6个单菌落中获得1个cpe+菌落,构成比为16.7%（1/6）;病例3未分离到cpe+菌落,病例4未分离到产气荚膜梭菌。11株产气荚膜梭菌菌株包括A型和C型两种,包含5种PFGE带型,分离自病例1和病例2的cpe+阳性菌株PFGE带型一致。结论 本次暴发事件可能由产气荚膜梭菌导致,综合使用各种实验室检测方法可在产气荚膜梭菌诊断标准滞后的情况下协助暴发事件分析。     

产气荚膜梭菌TaqMan实时荧光定量PCR特异性引物设计和方法验证

目的 建立针对水样中产气荚膜梭菌检测的TaqMan实时荧光定量聚合酶链式反应(PCR)方法,并测试该方法在自来水样中的检测效果。方法 选择位于该菌拟核中高度保守的plc基因,设计特异性引物和TaqMan探针,经优化后建立了针对该菌的TaqMan实时荧光定量PCR检测方法,结合滤膜法处理含有plc基因的标准菌株的模拟污染水样,并对所建立的方法进行测试。结果 所建立的产气荚膜梭菌TaqMan实时荧光定量PCR检测方法具有高度的特异性,13株食源性致病菌、3株艰难梭菌及1株腐败梭菌的Ct值大于40;该方法的最低检出限为1×10 copies/μL,具有较高的灵敏性;对模拟污染水样的最低检测限为1.0×10² CFU/mL。应用该方法对4份人工模拟污染阳性水样与90份自来水样进行检测发现,2份1.0×10² CFU/mL的模拟污染水样可检出产气荚膜梭菌,2份1.0×10 CFU/mL的模拟污染水样与90份自来水样均未检出产气荚膜梭菌。结论 所建立的产气荚膜梭菌TaqMan实时荧光定量PCR检测方法具有特异性好、灵敏性高的优点,对水体中产气荚膜梭菌的检...     

Growth of Clostridium perfringens from spore inocula in cooked cured beef: development of a predictive model

The objective of this study was to develop a model to predict the growth of C. perfringens from spores at temperatures applicable to the cooling of cooked cured meat products. C. perfringens growth from spores was not observed at a temperature of 12 °C for up to 3 weeks. The two parameters: germination, outgrowth, and lag (GOL) time and exponential growth rate, EGR, were determined using a function derived from mechanistic and stochastic considerations and the observed relative growths at specified times. A general model to predict the amount of relative growth for arbitrary temperature was determined by fitting the exponential growth rates to a square root Ratkowsky function, and assuming a constant ratio of GOL and generation times. The predicted relative growth is sensitive to the value of this ratio. A closed form equation was developed that can be used to estimate the relative growth for a general cooling scenario and determine a standard error of the estimate. The equation depends upon microbiological assumptions of the effect of history of the GOL times for gradual changes in temperature. Applying multivariate statistical procedures, a confidence interval was computed on the prediction of the amount of growth for a given temperature. The model predicts, for example, a relative growth of 3.17 with an upper 95% confidence limit of 8.50 when cooling the product from 51 to 11 °C in 8 h, assuming a log linear decline in temperature with time.     

辽宁省婴幼儿食品源阪崎克罗诺杆菌生物膜与耐药性分析

目的 探究婴幼儿食品中的阪崎克罗诺杆菌在形成生物膜后对其耐药性表达的影响,并对该致病菌的耐药性监测提供建议。方法 本研究对2018—2021年辽宁省婴幼儿配方食品中分离出的58株阪崎克罗诺杆菌进行了15种抗生素的药物敏感性、生物膜成膜力检测和不同生物态下耐药性分析。结果 辽宁省婴幼儿食品中检出的阪崎克罗诺杆菌对头孢唑啉、头孢噻肟、复方磺胺、氯霉素、头孢西丁、萘啶酸、氨苄西林/舒巴坦存在不同程度耐药性,88%的受试菌株对多黏菌素的耐药性达到中介,并且存在多重耐药菌;婴幼儿奶粉源阪崎克罗诺杆菌的耐药性均高于婴幼儿谷物辅食源阪崎克罗诺杆菌;生物膜成膜率100%,中等黏附力菌株居多占61.02%;基于生物膜态对应受试菌的药敏试验结果分析,头孢唑啉、头孢噻肟、亚胺培南、四环素和氨苄西林的药物抗性均增加,复方磺胺的药物抗性消失。结论 辽宁省市售婴幼儿食品中阪崎克罗诺杆菌不仅对多种抗生素具有单一耐药性,且存在多重耐药株;不同生物态的阪崎克罗诺杆菌均对头孢类抗生素药物抗性较高,同时生物膜态可以改变其药物抗性,应持续对婴幼儿食品源中不同生物态的阪崎克罗诺杆菌进行耐药监测。     

辽宁省婴幼儿食品中肠集聚性大肠埃希菌的病原学特征及耐药谱研究

目的 对2018—2020年辽宁省婴幼儿食品中致泻大肠埃希菌开展持续监测,了解和掌握本省内致泻大肠埃希菌的病原学特征、药物敏感性特征,为本省致泻大肠埃希菌流行病学研究奠定坚实基础,为可能导致的食源性疾病合理用药提供依据。方法 采集辽宁省共208份婴幼儿食品,分离肠杆菌科细菌,并进行16S rRNA基因测序分析与生化鉴定。对鉴定得到的大肠埃希菌进行毒力基因检测,收集分离的肠集聚性大肠埃希菌（EAEC）进一步进行血清学分型和耐药谱研究。结果 16S rRNA基因测序通过对肠杆菌科进行种鉴定,发现与生化结果一致。其中EAEC的检出率较高,且EAEC的毒力基因pic携带率高。毒力基因分型和血清分型具有一致性。共检测出25株EAEC,其中40%菌株（血清型O134:H9）携带毒力基因pic,28%菌株（血清型O3:H2）同时携带毒力基因aggR和astA,16%菌株（血清型O9:H6）携带毒力基因aggR,16%菌株（血清型O62:H7）携带毒力基因astA。78株大肠埃希菌中EAEC毒力基因的携带率为32.1%。25株EAEC为多重耐药株,主要对β内酰胺类、大环内酯类、喹诺酮类、四环素类抗菌药...     

The fate of Clostridium perfringens spores exposed to ozone and/or mild heat pretreatment on beef surfaces followed by modified atmosphere packaging

Clostridium perfringens is a natural contaminant of raw beef products that can proliferate to dangerous cell levels under conditions of temperature abuse. Spores of the bacterium were inoculated onto irradiated London broil beef at levels of 3 log₁₀ spores/g beef. Samples of beef (7.5×10.0×1.0 cm) were treated with aqueous ozone (5 ppm O₃ for 5 min), or heat (60°C for 30 min), or both and then vacuum-packaged to 2 kPa for up to 10 d storage at 37°C, 25°C, or 4°C. Storage at 37°C resulted in increases in viable counts after 1 d to over 7 log₁₀ cfu/g beef, whereas storage at 4°C prevented spore germination and growth for all treatments. At 25°C, heat-treated beef samples reached 6 log₁₀ cfu/g viable counts in 2 d and spores/vegetative cells on control or ozone-treated samples did not germinate or grow through the first day of vacuum-packaged storage. Modified atmospheres with increasing CO₂ concentration were also compared with regard to bacterial survival during beef storage at 25°C. C. perfringens spores remained dormant in control and ozone-treated beef during a 10-d storage at 25°C. Pretreatment with heat increased germination and outgrowth during storage of beef, whereas ozone treatment and no treatment controls were effective in inhibiting spore germination and outgrowth in combination with increasing CO₂ concentrations above 30% or refrigeration. These data support the avoidance of heat in the pretreatment of raw beef.