食品中小肠结肠炎耶尔森氏菌检验方法的研究 Ⅰ肉中小肠结肠炎耶尔森氏菌分离方法的研究

小肠结肠炎耶尔森氏菌是一个近年来引起食物中毒的新型菌,现已从许多种食品中分离到了该菌。近年来国内外多采用在4℃经7,14、21天冷增菌后,用 CIN 耶尔森氏菌选择性培养基来分离小肠结肠炎耶尔森氏菌,同时,对污染较严重的肉类食品,采用分离前用弱碱处理以破坏大量杂菌,提高该菌的检出率国内所见报道还多采用MAC,S S 这两种常用的肠道分离培养基,;也有报道用改良 Y 选择性培养基进行分离的。根据我国现有条件,我们改变了 CIN 培养基中的一些成分,命名为 CIN-I;比较小     

RF-LAMP技术检测鸡肉中小肠结肠炎耶尔森氏菌

本研究建立实时荧光环介导等温扩增技术检测小肠结肠炎耶尔森氏菌的方法,扩增产物经电泳和酶切鉴定,表明扩增结果正确。本研究验证该方法特异性,对灵敏度和人工污染鸡肉的检出限进行测定。结果表明小肠结肠炎耶尔森氏菌株呈阳性结果,非小肠结肠炎耶尔森氏菌株均呈阴性结果。该方法检测纯菌的灵敏度为61 CFU/m L,人工污染鸡肉的检出限为46 CFU/g。该方法特异性强、灵敏度高、可实时检测小肠结肠炎耶尔森氏菌,能够实现对小肠结肠炎耶尔森氏菌的快速检测。     

小肠结肠炎耶尔森氏菌分子生物学检测技术研究进展

小肠结肠炎耶尔森氏菌（Yersinia enterocolitica,Y. enterocolitica）是一种重要的食源性致病菌,可以引起人类的耶尔森氏菌病,该病的典型症状为腹泻、回肠炎和肠系膜淋巴结炎等。由于该菌在特定食品（如肉与肉制品）中的检出率较高,因此对食品安全构成一定的威胁。就现有的检测方法而言,分离纯化和生理生化鉴定方法耗时且难以检测出复杂食品基质中低浓度的小肠结肠炎耶尔森氏菌。所以,研发能够快速、准确地检测食品中小肠结肠炎耶尔森氏菌的鉴别方法迫在眉睫。该研究将小肠结肠炎耶尔森氏菌现有的分子生物学检测方法分为两大类：变温扩增技术和等温扩增技术进行相关研究最新进展的总结,并对其优缺点和发展方向进行讨论,以期为小肠结肠炎耶尔森氏菌快速检测方法研究提供参考。     

新等温扩增技术检测小肠结肠炎耶尔森氏菌

应用新型等温扩增检测技术——交叉引物等温扩增结合免疫金标试纸条建立检测小肠结肠炎耶尔森氏菌的方法。针对小肠结肠耶尔森氏菌16-23S rDNA间区序列设计特异性引物及探针,用54株小肠结肠炎耶尔森氏菌及相近株细菌进行特异性试验;通过纯菌液计数、样品中添菌检测进行灵敏度验证;对677份食品用传统生化国标法进行比较检测试验。建立方法具有较好特异性;增菌液检测灵敏度为10~1cfu/mL,当每25g样品中有10~0cfu菌时经增菌步骤后即可检出,样品检测同传统检测结果比较大致相符,没有漏检,假阳性率较低。建立的新型恒温检测方法可用于食品中小肠结肠炎耶尔森氏菌初筛检测。     

食品中小肠结肠炎耶尔森氏菌分离鉴定方法研究进展

小肠结肠炎耶尔森氏菌是一种具有嗜冷特性的食源性致病菌,广泛存在于食品中,对人们健康构成较大威胁。建立高效的分离鉴定技术对该菌进行监控是相关食品安全的重要保障。本文对小肠结肠炎耶尔森氏菌的分离过程,包括分离前增菌、分离前碱处理和平板分离,以及传统生化和血清型鉴定、分子生物学鉴定、免疫学鉴定和谱学鉴定方法等方面的研究进展进行了阐述。小肠结肠炎耶尔森氏菌的深入检测研究不仅有赖于现有分离鉴定方法的不断优化,还需要开发更多新型检测技术应用于菌株溯源分析、流行病学和耐药性等方面的研究,以期为该类菌株资源数据库的建立和整理,预防相关耶尔森氏菌疾病的爆发提供良好的保障。     

食品中小肠结肠炎耶尔森氏菌检验方法的研究 Ⅱ、乳及其制品中小肠结肠炎耶尔森氏菌分离方法的研究

小肠结肠炎耶尔森氏菌主要存在于肉、乳及其制品这两大类食品中;在《食品中小肠结肠炎耶尔森氏菌检验方法的研究》的第一部分,我们研究了从肉及其制品中分离该菌的检验方法,比较了冷增菌和碱处理方式对该菌分离效果的影响,证实了冷增菌和碱处理可以提高该菌的检出率;同时根据国际上通用的 CIN 耶尔森氏菌选择性培养基推出了 CIN-I 培养基,比较了 CIN-I,MAC、SS 和改良 Y 4种分离培养基的分离选择效果。在这部分里,我们将研究冷增菌和碱处     

环介导等温扩增技术检测小肠结肠炎耶尔森氏菌的研究

采用环介导等温扩增技术(Loop-mediated isothermal amplification,LAMP)快速检测小肠结肠炎耶尔森氏菌(Yersinia enterocolitica)。以小肠结肠炎耶尔森氏菌(AY004311.1)Ail基因序列作为靶序列,设计内、外引物,通过肉眼观察沉淀判断检测结果。结果表明:LAMP检测小肠结肠炎耶尔森氏菌的灵敏度为6.3cfu/mL,人工污染鸡肉的检出限为340cfu/g。PCR检测小肠结肠炎耶尔森氏菌的灵敏度为630cfu/mL,人工污染鸡肉的检出限为3.4×104cfu/g。采用试剂盒法提取DNA,从样品处理到报告结果,LAMP方法耗时2h,PCR方法耗时3h。因此,LAMP检测小肠结肠炎耶尔森氏菌的灵敏度高,耗时短,特异性好,操作简便,无需特殊的仪器设备,适合在我国广大基层实验室开展应用,为快速检测食源性致病菌构建了一个新的技术平台。     

小肠结肠炎耶尔森氏菌PCR反应体系的快速优化

影响PCR反应结果的因素较为复杂．为了快速、准确地得到PCR反应的最适条件，避免假阴性或假阳性结果的发生，在研究小肠结肠炎耶尔森氏菌PCR反应体系优化中，采用了正交试验法．通过设计两组正交试验，准确地找出了检测小肠结肠炎耶尔森氏菌的最佳扩增条件，结果表明该方法高效快速，是PCR反应体系优化中值得推广使用的方法。     

辅酶Q0对小肠结肠炎耶尔森氏菌的抑杀作用及对菌体细胞膜的影响

小肠结肠炎耶尔森氏菌是一种广泛分布于蔬菜、乳制品、肉类、豆制品和沙拉中的食源性致病菌，该菌引起的耶尔森氏菌病属于较严重的人畜共患病，对公共卫生构成重大威胁。本文探究辅酶Q0对小肠结肠炎耶尔森氏菌的抑杀作用及对菌体细胞膜的影响，利用液体稀释法测定辅酶Q0对小肠结肠炎耶尔森氏菌的最小抑菌浓度(MIC)，随后，依据MIC的试验结果测定最小杀菌浓度(MBC)，以及对菌体生长曲线的影响，通过检测辅酶Q0对小肠结肠炎耶尔森氏菌在LB介质中的抑杀作用，评价其抑菌效果。通过测定辅酶Q0对小肠结肠炎耶尔森氏菌膜电位、胞内ATP浓度、细胞膜完整性以及菌体细胞形态的影响，探究其对菌体细胞膜的影响。试验测得辅酶Q0对小肠结肠炎耶尔森氏菌ATCC 23715的MIC和MBC均为0.10 mg/mL，辅酶Q0作用后使菌体生长延滞期延长，最大生长速率减小。探究辅酶Q0的抑杀作用，发现当辅酶Q0质量浓度为MIC时，细菌总数于6 h后降至检出限以下；当质量浓度为2 MIC时，细菌总数于3 h后降至检出限以下。辅酶Q0处理使菌体细胞膜电位显示超极化，胞内ATP浓度降低，细胞膜完整性降低，细胞形态出现凹陷、皱缩现象，表明菌体细胞膜完整性和通透性发生改变。本研究结果表明:辅酶Q0对小肠结肠炎耶尔森氏菌ATCC 23715表现出良好的抑杀效果，其抑菌作用通过影响细胞膜的完整性和通透性实现，研究结果为辅酶Q0用于食品工业中控制小肠结肠炎耶尔森氏菌提供理论依据。     

鲜肉中小肠结肠炎耶尔森菌的半定量风险评估

目的:分析南京市售鲜肉中小肠结肠炎耶尔森菌的风险程度。方法:将国内外文献报道及市场调查数据的统计分析相结合,运用经典的半定量风险评估软件"Risk Ranger"进行风险分级。结果:南京市售猪肉和牛肉中小肠结肠炎耶尔森菌的风险评分分别为31和24,每人每天因食用污染该菌的鲜肉造成食物中毒的概率分别为3.62×10-9,3.12×10-10,前者为后者风险的11.6倍。通过进一步研究发现,加工后采取有效的控制体系,均使两者的风险均降低到10%。结论:南京市售牛肉中小肠结肠炎耶尔森菌的风险程度较低,而市售猪肉中已接近中等程度,需做好预防措施,加强监管。     

Isolation of Yersinia enterocolitica from foods.

Many selective enrichment and plating media for the isolation of Yersinia enterocolitica from foods are described. However, at present no single isolation procedure is available for the recovery of all pathogenic strains of Yersinia enterocolitica. Cold enrichment in phosphate-buffered saline plus 1% sorbitol and 0.15% bile salts (PBSSB) and two-step enrichment with tryptone soy broth (TSB) and bile oxalate sorbose (BOS) broth are very efficient methods for the recovery of a wide spectrum of serotypes of Y. enterocolitica. Enrichment in irgasan ticarcillin chlorate (ITC) broth was found to be the most efficient method for the recovery of strains of serotype 0:3, which is the most common clinical serotype of Y. enterocolitica in Europe. Post-enrichment alkali treatment often results in higher isolation rates. Cefsulodin irgasan novobiocin (CIN) agar and Salmonella-Shigella deoxycholate calcium chloride (SSDC) agar are the most commonly used plating media. For the recovery of serotype 0:8 strains, the common clinical isolates in North America, enrichment in BOS and plating on CIN seems the most efficient procedure. Selection of the proper enrichment procedure will depend on the bio/serotypes of Yersinia spp. sought and on the type of food to be examined. The use of more than one medium for both enrichment and plating will result in higher recovery rates of Yersinia spp. from foods. Parallel use of the following two isolation procedures is recommended. (1) Enrichment in ITC for 2 days at 24 degrees C; plating on SSDC agar (2 days at 30 degrees C). (2) Pre-enrichment in TSB for 1 day at 24 degrees C; enrichment in BOS for 5 days at 24 degrees C; alkali treatment (mixing 0.5 ml enriched broth with 4.5 ml of 0.5% KOH in 0.5% NaCl for 5 s); plating on CIN agar (2 days at 24 degrees C).     

Comparison of the chlorine inactivation of Yersinia enterocolitica in chlorine demand and demand-free systems

Inactivation of Yersinia enterocolitica by chlorine (0.6 to 20 ppm) was investigated in distilled water and in tryptic soy broth (TSB, 0.015%) at different temperatures (4, 20, and 40 degrees C). In distilled water, chlorine inactivation of Y. enterocolitica was enhanced by increasing the temperature from 4 to 20 degrees C, and survival curves were described by a model that assumed first-order kinetics followed by tailing in which the microbial concentration remained constant. The presence of TSB increased chlorine resistance of Y. enterocolitica, and survival curves were concave downward. These survival curves were described by a model based on the Weibull distribution. Chlorine decay in distilled water was independent of temperature and of the initial concentration of available chlorine and was modeled by first-order reaction kinetics. Chlorine decay in TSB was independent of the initial chlorine concentration but depended on the treatment temperature and was modeled by the addition of two first-order decay equations. The increased resistance of Y. enterocolitica to chlorine in TSB was not due only to the chlorine demand by the TSB components. These components protected Y. enterocolitica cells from the antimicrobial effect of chlorine.     

Comparison of four methods for isolation of Yersinia enterocolitica from raw and pasteurized milk from northern Iran

Four methods for isolation of Yersinia enterocolitica from raw and pasteurized milk from northern Iran were compared. Three hundred and ten raw milk samples were collected from tanks on their arrival at various central dairies, and 40 pasteurized milk samples were collected from tanks on their arrival at a manufacturing plant. Each sample was examined for the presence of Y. enterocolitica by (1) direct culture; (2) enrichment in double-strength buffered peptone water at 4 degrees C for 1 month; (3) enrichment in modified Rappaport medium at room temperature for 72 h after a preenrichment in double-strength peptone water at 4 degrees C for 1 month; and (4) enrichment in a medium containing sucrose, tris (hydroxymethyl) aminomethane, sodium azide, and ampicillin at 28 degrees C for 48 h after a preenrichment in double-strength peptone water at 4 degrees C for 1 month. All samples and enrichments were spread on MacConkey agar plus calcium chloride and Tween 80, Yersinia selective agar, and Hektoen medium plus ampicillin. Five samples (1.6%) of raw milk but no pasteurized milk samples were positive for Y. enterocolitica. No Y. enterocolitica were recovered by methods 1 or 2. Y. enterocolitica were recovered from 2 samples by method 3 followed by culture on Yersinia selective agar, and from 5 samples by method 4 followed by culture on Hektoen medium plus ampicillin. The isolates were biotype 1A or 1B, serotype O:7-13 or O:9 and phage type Xo or Xz. All isolates were resistant to ampicillin and amoxicillin, and sensitive to tetracycline, streptomycin, chloramphenicol, and trimethoprim-sulfamethoxazole.     

Evaluation of selective enrichment PCR procedures for Yersinia enterocolitica

Four enrichment PCR protocols for detecting unlysed cells of pathogenic Yersinia enterocolitica were studied. First, the probability of detecting Y. enterocolitica cells of known concentrations by a multiplex PCR assay was determined, and it was found to follow a logistic regression model. From this model, the probability of detecting Y enterocolitica at a specific concentration could be estimated; for example, the detection probability of 10(4) CFU/ml was estimated to be 85.4%. The protocols were evaluated on enrichment cultures inoculated with 10(2) CFU/ml Y. enterocolitica and 10(2)-10(6) CFU/ml of a defined background flora. For each protocol, the time for sample withdrawal and the presence of background flora were studied with respect to PCR detection. The optimal point in time of sample withdrawal was found to be different for each protocol employed. Early detection was favoured by concentrating the target cells, and the most rapid PCR detection of Y. enterocolitica was achieved with enrichment in Yersinia-PCR-compatible-enrichment (YPCE) medium for 3 h at 25 degrees C, followed by a centrifugation prior to PCR analysis. For detection of Y. enterocolitica in the presence of high concentrations (10(6) CFU/ml) of background flora, a long incubation time followed by density centrifugation and a dilution step was most successful. The protocol that gave the most reliable PCR detection in the presence of 10(6) CFU/ml background flora included 24 h incubation in Yersinia-selective-enrichment (YSE) broth at 25 degrees C, followed by Percoll density centrifugation, and a 100 times dilution prior to PCR analysis.     

Detection, isolation and enumeration of Yersinia enterocolitica from raw pork

The methods available for the isolation of Yersinia enterocolitica from foods are generally considered to be less than optimal, and methods for estimation of numbers are lacking. Such methods are needed to understand better the significance of foodborne yersiniosis and to provide data for exposure assessment. We describe a method for the detection and enumeration of Y. enterocolitica containing the pYV virulence plasmid (YeP+) in samples from pork surfaces. The method uses a multiplex PCR targeting the ail and virF genes to detect Y. enterocolitica after incubation of surface swabs in Yersinia enrichment broth according to Ossmer. Enumeration was achieved by adapting the enrichment to a most probable number (MPN) method format. A presumptive result was available within 24 h of sample receipt, and YeP+ isolates were confirmed within four days. The presence/absence and MPN methods were evaluated in a pilot survey of 34 packs of raw pork meat purchased from retail outlets in Christchurch, New Zealand. YeP+ was detected by PCR on meat from 32% of the packs, and YeP+ isolates were obtained from 18% of the samples. YeP+ were present at numbers ranging from 0.30 to 5.42 MPN/cm(2). This improved method for the detection and enumeration of YeP+ from meat samples can be used for microbiological surveys to obtain data for assessments of consumer exposure to virulent Y. enterocolitica, and in outbreak investigations.     

Enrichment procedures and plating media for isolation of Yersinia enterocolitica

A shortened enrichment procedure (25 degrees C for 24 h) was compared with cold enrichment procedures (4 degrees C for 1 to 3 weeks) and direct plating for isolation of Yersinia enterocolitica from commercial ground meat samples. The combined data of all recovery procedures showed that this organism was isolated from 34% of the ground beef samples. The highest isolation rate was 32% for the 4 degrees C/3-week enrichment, followed by 28% for the 4 degrees C/2-week enrichment, 26% for the 25 degrees C/24-h enrichment, 22% for the 4 degrees C/1-week enrichment, and 10% for direct plating. No significant differences (P > 0.05) in isolation rate occurred between the 4 degrees C/3-week, 4 degrees C/2-week, 25 degrees C/24-h, and 4 degrees C/1-week enrichments. The combined data of all recovery procedures showed that Y. enterocolitica was isolated from 64% of ground pork samples. The highest isolation rate was 48% for the 4 degrees C/3-week enrichment, followed by 40% for the 25 degrees C/24-h enrichment, 34% for the 4 degrees C/2-week enrichment, 24% for the 4 degrees C/1-week enrichment, and 24% for direct plating. No significant differences (P > 0.05) in isolation rate occurred between the 4 degrees C/3-week, 25 degrees C/24-h, and 4 degrees C/2-week enrichments. During the plating phase of the experiment, the efficiency of a dye-containing, Yersinia-selective medium (KV202) was compared with that of a commercially available cefsulodin-irgasan-novobiocin medium. Recovery rates were similar for both media. However, KV202 agar differentiated Y. enterocolitica from such contaminating bacteria as Enterobacter, Serratia, and Salmonella by colony morphologic characteristics and color.     

Use of a culture-independent molecular method to study the ecology of Yersinia spp. in food

A culture-independent method for the direct detection in food of Yersinia spp. was developed in this study. It is based on the amplification of a 359 bp PCR product from the RNA polymerase beta-subunit gene (rpoB) and subsequent analysis by denaturing gradient gel electrophoresis (DGGE). Direct detection of Yersinia spp. by PCR-DGGE was carried out in ready-to-eat vegetables and the results compared with the results of the traditional, culture-dependent method. The DGGE profiles were determined to be species-specific. As a matter of fact, Yersinia enterocolitica, Yersinia intermedia, Yersinia frederiskenii and Yersinia kristensenii showed differential migrations in the gels. Moreover, Y. enterocolitica serotypes O:3, O:5 and O:9 were distinguishable, as well. Only for a limited number of traditionally isolated strains, the biochemical and molecular identification agree. In particular, an overestimation of Y. enterocolitica, as determined biochemically, was observed. Finally when the protocol was applied to 27 food samples, a good correlation was obtained when the results of traditional and direct methods were analyzed. The molecular method was able to identify Y. enterocolitica, not detected by plating analysis. However, for 4 samples, that, by plating analysis, were determined to contain Yersinia spp., no PCR product could be obtained after enrichment, probably due to low numbers of target cells, thereby not allowing the possibility to perform DGGE analysis. The protocol described here represents a reliable tool for the detection of Yersinia spp. in food, which can be used to obtain the needed results faster than with traditional culturing methods.     

Evaluation of a 5'-nuclease (TaqMan) assay with the thin agar layer oxyrase method for the detection of Yersinia enterocolitica in ground pork samples

A 5'-nuclease (TaqMan) assay was evaluated for its capability to recover and detect stressed Yersinia enterocolitica. Sensitivity studies of a 5'-nuclease assay for detecting Y. enterocolitica 0:8 in a pure culture system and spiked ground pork samples demonstrated that the assay has reliable sensitivity with a detection limit of 3 to 4 log CFU/ml or CFU/g. The PCR 5'-nuclease (TaqMan) assay was evaluated with the Thin Agar Layer Oxyrase method (TALO, overlaying 14 ml of Trypticase soy agar with a 1:30 dilution of "Oxyrase for Agar" onto a prepoured pathogen-specific, selective medium), and it was compared against the selective medium cefsulodin-irgasan-novobiocin (CIN) for recovering and detecting Y. enterocolitica from inoculated nonfrozen and frozen (-15 degrees C, 2 days) ground pork samples. The TALO method showed more sensitivity (detection limit, 2 log CFU/ml), and it has greater recovery capability (0.5 to 1 log CFU/ml) than CIN (P < 0.05). The 5'-nuclease assay provided rapid detection processing (5 versus 24 h after an 18-h enrichment). The sensitivity per PCR was calculated to as low as 0 to 1 log CFU per PCR reaction; however, in the method's current developmental stage, target pathogens should be enriched to 3 to 4 log CFU/ml or CFU/g to show consistent results. In a survey of 100 ground pork samples using TALO, CIN, and PCR methods, no Y. enterocolitica was recovered. A combined cultivation and an automated PCR TaqMan could be used as a presumptive screening test for detecting Y. enterocolitica in food samples.     

Yersinia enterocolitica biogroup 1A, serotype O:5 in chicken carcasses

To evaluate the presence of Yersinia enterocolitica in raw chicken, 70 samples of chicken carcasses were obtained from a Buenos Aires, Argentina, processing plant. The detection of this psychotropic microorganism was carried out using the whole carcass rinse method and enrichment in phosphate-buffered saline, 0.067 M, pH 7.6, at 4 degrees C, followed by alkaline treatment and isolation on cefsulodin-irgasan-novobiocin nutrient agar. Y. enterocolitica or related species were detected in 7 of 70 samples. From them, 4.3% were identified as Y. enterocolitica, 1.4% as Yersinia intermedia, and 4.3% as Yersinia frederiksenii. The serotype and phagotype of Y. intermedia isolates were O:5(2), 5(3), and Xz and those of Y. frederiksenii were O:4.16 and Xo. All Y. enterocolitica isolates belong to the biogroup 1A, serotype O:5, phagotype Xz, which presents uncertain pathogenic potential. These findings reinforce the worldwide concern on the microbiological quality of food products. Quality assurance programs on the whole poultry process are increasingly being adopted in Argentina.     

2018—2020年高邮市小肠结肠炎耶尔森菌流行病学及病原学特征分析

目的 了解高邮市小肠结肠炎耶尔森菌流行病学及病原学特征,为本地区该菌的防控提供科学依据。方法 2018-2020年在高邮市采集腹泻患者粪便、家畜家禽粪便、苍蝇及生猪屠宰场组织样本等各类样本共计2682份。采用冷增菌方法进行小肠结肠炎耶尔森菌的分离培养,对分离菌株进行生化鉴定、血清分型、生物分型和毒力基因的荧光PCR检测。结果 2682份样本中共检出小肠结肠炎耶尔森菌120株,检出率为4.47%,包括猪粪源菌株99株,鸭粪源菌株2株,苍蝇源菌株4株,生猪屠宰场源菌株15株。分离菌株血清型以O:3型为主,生物型主要为3型,毒力基因检测结果显示,具有致病性的分离菌株共73株,其中O:3血清型69株、O:9血清型1株和血清未定型3株,且以猪粪源菌株为主。结论 小肠结肠炎耶尔森菌在高邮市畜禽动物、苍蝇媒介及生猪屠宰场中存在流行分布,猪的检出率最高,且致病性菌株携带率最高,主要致病型别为生物3型/ O:3血清型（3/ O:3型）。