整鸡中弯曲菌定量检测方法的建立与优化

目的建立一种简便、灵敏、稳定的整鸡中弯曲菌定量检测方法,并用北京地区市售整鸡样品进行验证。方法分别对37株鸡肉中常污染的背景杂菌、24株弯曲菌菌株进行抗生素敏感性测试,对弯曲菌分离用选择性培养基及抗生素添加剂进行优化和改进;用77份人工定量污染弯曲菌的鸡淋洗液进行添加回收实验,以生长指数评价选择性平板对鸡肉样品中背景杂菌的抑制情况和弯曲菌生长状况影响。结果在改良后的Karmali和Preston选择性平板上,弯曲菌生长状态稳定,并可有效抑制鸡肉样品中常见的背景干扰杂菌的生长繁殖,此方法对鸡肉中弯曲菌检出的灵敏度可达2.5 CFU/g。结论所建方法灵敏度高、操作简便、计数准确,Karmali和Preston平板组合后可有效提高鸡肉中弯曲菌的检出率,适用于禽类食品中弯曲菌的定量检测。     

扬州市市售鸡肉中弯曲菌定性定量流行病学分析

目的调查分析超市和农贸市场零售鸡肉中弯曲菌定量污染水平,为弯曲菌污染的定量风险评估、危害分析和防控提供数据。方法选择扬州市内各大型超市和农贸市场作为采样点,采取未分割的鸡胴体,在3 h内送到实验室开展检测。运用选择性CCDA平板直接计数法对弯曲菌进行定量检测,涂布平板法计数,用PCR进行鉴定。结果采集的40只整鸡,检测出阳性样品19份,平均阳性率为47.5%,平均带菌量为229.2 CFU/g。其中超市的8份阳性整鸡中,平均带菌量为28.3 CFU/g;农贸市场的11份阳性整鸡中,平均带菌量为313.6CFU/g。共分离出56株弯曲菌,其中空肠弯曲菌29株,结肠弯曲菌23株,其他弯曲菌4株。结论定性及定量结果显示,零售鸡中存在弯曲菌污染,农贸市场的零售鸡肉比超市的零售鸡肉污染严重,需要加强防控措施。     

含内标的多重实时PCR法同时检测空肠弯曲菌和结肠弯曲菌

目的建立含内标的多重实时荧光PCR法同时检测空肠弯曲菌和结肠弯曲菌。方法针对空肠弯曲菌特有hipO基因和结肠弯曲菌特有ceuE基因设计引物探针,设计并优化内标DNA添加量。测试了方法的特异性、灵敏度以及在鸡肉中的检出限。结果内标的最适添加量为10~4copies/PCR。所建立方法对空肠弯曲菌和结肠弯曲菌的灵敏度分别达到4.7copies/PCR和5.23copies/PCR;对115株空肠弯曲菌、49株结肠弯曲菌和42株非目标菌株在3种不同类型的实时荧光PCR仪上的特异性均达到100%;对鸡肉中空肠弯曲菌和结肠弯曲菌的检出限达到10CFU/25g,与传统检测方法一致。采用所建立的方法对50份市售生鲜鸡肉进行检测发现,空肠弯曲菌阳性率为12%(6/50),结肠弯曲菌阳性率为4%(2/50);传统国标检测方法除了1份空肠弯曲菌阳性样品未得到分离确认,其余PCR阳性样品均在平板上分离确认。结论该方法特异性强、灵敏度高、开放性好、含有内标可防止"假阴性",可应用于食品中2种重要致病性弯曲菌的快速同步检测。     

猪肉屠宰加工过程中弯曲菌流行病学调查与分析

目的 调查分析猪肉屠宰加工过程中主要环节弯曲菌的污染现状。方法 选择两家猪肉屠宰加工企业,分别采集宰前、脱毛、取内脏、胴体修饰和预冷前后等环节样品,应用选择性CCDA平板直接计数法对弯曲菌进行定量检测。结果 150份猪肉样品检测出22份弯曲菌阳性样品,阳性率14.67%;环境样品30份,检出12份阳性样品,阳性率40.00%。所检出的弯曲菌均为结肠弯曲菌,未见其它种属弯曲菌。所监测的6个环节均存在不同程度的弯曲菌污染,阳性率呈现先升高后降低的变化趋势。其中取内脏环节弯曲菌阳性率最高,为17.50%,阳性样品弯曲菌携带量为177.14±296.73CFU/100cm2;而环境样品中取内脏环节的地面样品阳性率最高,为100%,弯曲菌携带量为588.00±307.66CFU/100cm2。结论 猪肉屠宰加工过程中存在弯曲菌污染,以取内脏环节阳性率最高,预冷后阳性率明显降低;而环境样品的高污染率对猪肉屠宰加工过程中的交叉污染构成了很大威胁。     

mini-MPN-STE-qPCR法快速定量检测食品中沙门氏菌

目的 建立一种快速、简易、可定量检测食源性沙门氏菌定量PCR(quantitative PCR, qPCR)方法。方法 依据沙门菌属invA基因序列设计染料法qPCR引物, 建立基于嵌合荧光法的沙门氏菌qPCR检测方法, 并通过短时间增菌(short time enrichment, STE)的5管微型多管发酵计数法（mini-most probable number, mini-MPN）进行定量检测, 构建mini-MPN-STE-qPCR法。使用人工污染的鸡肉混合液进行定量检测, 检测结果与传统MPN计数法和平板计数法进行比较分析。结果 建立的 qPCR法特异性良好, 方法灵敏度为50 CFU/mL, 结合48孔板min-MPN和4h 短时间增菌建立的mini-MPN-STE-qPCR法可将整个检测流程缩短至7 h。人工添加沙门氏菌的鸡肉混合液检测结果表明方法检出限为-0.347 log MPN/mL, Bland-Altman分析结果显示, 此法与传统MPN计数法相关系数R2=0.994, 与平板计数法相关系数R2=0.992。结论 该方法准确、简单易行、成本低、灵敏度高, 可用于食品中沙门菌的快速定量检测。     

基于实时荧光环介导等温扩增快速检测鸡肉中的大肠杆菌O157:H

以大肠杆菌O157:H7的VT2基因序列设计特异性引物,利用Midori Green新型核酸荧光染料,建立鸡肉中大肠杆菌O157:H7实时荧光定量环介导等温扩增(Rti-LAMP)检测方法。纯培养大肠杆菌O157:H7检测灵敏度达3.5 CFU/反应,需时45 min。模拟大肠杆菌O157:H7污染鸡肉样品,经37℃增菌4 h,用Whirl-pak无菌袋过滤离心得沉淀,提取样品DNA模板用于Rti-LAMP反应,检测大肠杆菌O157:H7灵敏度达140 CFU/g,整个检测流程约7 h。采用蒙脱石封闭的活性炭前处理污染鸡肉样品,不经增菌过程,结果表明:该Rti-LAMP检测方法灵敏度达12 CFU/g,整个检测耗时约4.5 h。市购鸡肉样品51份,以大肠杆菌国标检测法为对照,研究基于Rti-LAMP联合增菌或封闭活性炭预处理而不经增菌的两种方法,对比检测临床鸡肉样品大肠杆菌O157:H7污染率。结果:国标法和增菌的Rti-LAMP两种方法均检测到同一鸡肉样品为大肠杆菌O157:H7阳性,经封闭活性炭预处理而未经增菌的Rti-LAMP则检测到8份鸡肉样品大肠杆菌O157:H7阳性。研究表明,封闭活性炭预处理的Rti-LAMP检测方法较国标法更灵敏、快速、简便、特异地检测鸡肉中大肠杆菌O157:H7污染。     

涂布平板计数法与MPN法定量检测禽肉和蛋中沙门菌的比较

为选择良好的禽肉和鸡蛋中沙门菌定量检测方法 ,通过染菌实验 ,分析比较了涂布平板计数法和MPN法 ,结果表明平板计数法优于MPN法。应用平板计数法检测了 16份鸡蛋和 2 6份禽肉试样。沙门菌检出限分别为 1CFU ml和 10 0CFU g。 16份蛋类试样中均未检出沙门菌。 2 6份鸡肉试样中 ,2 0份为沙门菌阳性 ,阳性率为 77%。其中 9份试样沙门菌数低于检出限 10 0CFU g ,其余试样菌数范围为 1 5× 10 2 ～ 2 5× 10 5CFU g。对 3份 - 2 0℃保存 5d的阳性禽肉试样应用MPN法进行了冷冻前后沙门菌含量的比较分析 ,储存后的菌数比冷冻前略有增加。实验结果表明 ,在食物试样中杂菌污染严重或沙门菌水平较低的情况下 ,平板法不能准确进行沙门菌计数 ,仍需进行MPN定量检测。     

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。     

新型阪崎肠杆菌选择性分离培养基的研制

目的:筛选一种新型、特异性高的阪崎肠杆菌选择性培养基.方法:通过药敏实验,筛选出对阪崎肠杆菌耐药而对其他杂菌敏感的抗生素;利用抑菌浓度实验、抗生素组合平板实验,筛选出最佳的抗生素组合方式及浓度.将抗生素组合与显色剂添加到基础培养基中,配制成阪崎肠杆菌的新型选择培养基.结果:组合抗生素(4μg/mL万古霉素、0.1μg/mL头孢噻吩)和显色剂(0.1g/L 5-溴-4氯-3-吲哚-a-葡萄糖苷)添加到脑心培养基中,制成阪崎肠杆菌VC选择性显色培养基,能最大限度地抑制杂菌生长,而对阪崎肠杆菌的生长没有影响.经灵敏性实验,对接种的奶粉样品中的阪崎肠杆菌的检出限达到lcfu.结论:制得的新型阪崎肠杆菌选择性培养基特异性强、灵敏度高,可用于奶粉样品的分离检测.     

甲型副伤寒沙门菌特异性基因筛选及PCR检测体系建立

以甲型副伤寒沙门菌为检测目标,通过比较基因组和聚合酶链式反应（polymerase chain reaction,PCR）验证方法筛选到4 个该血清型的特异性基因,其中以gene_3105作为该血清型的检测靶点设计引物PA23;并结合沙门菌属特异性引物139-141,建立一种甲型副伤寒沙门菌的PCR检测方法。优化PCR反应体系,并对该检测体系的特异性、灵敏度、抗干扰能力及人工污染样品检出限等方面进行评价。结果表明,当样品中含有甲型副伤寒沙门菌时,该体系能扩增出2?条特异性条带,含有其他血清型的沙门菌仅能扩增出284?bp条带,不含沙门菌无扩增条带产生。灵敏度评价表明,基因组DNA和纯菌菌落检出限分别为32.4?pg/μL和4.3×103?CFU/mL;抗干扰能力实验显示,当鸡肉背景菌群和猪肉背景菌群浓度在106?CFU/mL和4.87×107?CFU/mL时,检出限为6.43×104?CFU/mL。当无菌的鸡肉和猪肉样品中添加N?CFU/25?g甲型副伤寒沙门菌时,经10?h增菌,检测结果为阳性（0＜N＜10）。实验建立甲型副伤寒沙门菌PCR检测方法具有较好的特异性和灵敏度,有很好的应用价值,可在食品安全领域广泛应用。     

Comparison of three methods for detecting Campylobacter spp. in chilled or frozen meat

There is a demand from the meat industry as well as from public health authorities for a simple and rapid detection method for thermophilic Campylobacter spp. from food. Hence, we compared different isolation procedures for their usefulness for this purpose. Bolton enrichment medium without blood, incubated statically in stomacher bags in microaerophilic atmosphere, detected more samples positive for thermophilic Campylobacter spp. than did Preston enrichment broth in bottles with small headspace and tight caps, incubated in aerobic atmosphere. Use of an automated antigen detection system to identify enrichment cultures positive for Campylobacter spp. was as sensitive as selective agars, and reduced the detection time by 24 h. Campylobacter spp. were recovered from 18.4% of the 461 samples tested. The prevalence was highest in refrigerated poultry meat (52% of the 80 samples tested) and poultry offal (41% of the 44 samples tested).     

Processing practices contributing to Campylobacter contamination in Belgian chicken meat preparations

The aim of this study was to obtain insight into processing practices in the poultry sector contributing to the variability in Campylobacter contamination in Belgian chicken meat preparations. This was achieved by company profiling of eleven food business operators, in order to evaluate variation of processing management, in addition to statistical modelling of microbiological testing results for Campylobacter spp. contamination in 656 end product samples. Almost half (48%) of chicken meat preparation samples were positive for Campylobacter spp. Results revealed a statistically significant variation in Campylobacter contamination between 11 chicken meat producers across Belgium at both quantitative and qualitative detection levels. All producers provided Campylobacter-positive samples, but prevalence ranged from 9% up to 85% at single producer level. The presence or addition of skin during production of chicken meat preparations resulted in almost 2.2-fold increase in the probability of a sample being positive for Campylobacter, while chicken meat preparations made from frozen meat, or partly containing pre-frozen meat, had a significant (Odds Ratio=0.41; CI 95% 0.18:0.98) lower probability of being positive for Campylobacter. However, the quantitative results indicated that the positive freezing effect on Campylobacter count was compromised by the presence and/or adding of skin.     

Isolation and identification of enteropathogenic Campylobacter spp. from chicken samples in Taipei

A total of 95 chicken samples that consisted of 34 whole chickens, 32 organs (gizzards and livers), and 29 chicken parts (drumsticks, wings, and breasts), collected from traditional retail markets (no chilling facilities) and supermarkets in Taipei, were examined for the occurrence of enteropathogenic campylobacters. Three selective media, Peterz's charcoal cefoperazone deoxycholate agar, Campy-Cefex agar, and charcoal-based selective medium, were evaluated for their efficacy to isolate Campylobacter spp. from chicken samples. The results showed that there were no differences among the three media to isolate Campylobacter spp. from all chicken samples (P > 0.05). However, there were markedly different isolation rates of campylobacters between supermarket and retail market (P < 0.05). Enteropathogenic campylobacters (C. jejuni and C. coli) were found on 68% of whole chickens, 100% of chicken parts, and 100% of organs from retail markets. In supermarkets, the isolation rates of these campylobacters from whole chickens, chicken parts, and organs were 42%, 53%, and 60%, respectively. The low isolation rates of the two campylobacters isolated from chicken samples in supermarkets differed statistically from those obtained from traditional retail markets (P < 0.10). The API CAMPY test kit also was evaluated for the identification of Campylobacter spp. as compared with the conventional identification method. The results showed that the API CAMPY test kit (Biomerieux, Marcy-l'Etoile, France) could efficiently detect 87 Campylobacter spp. isolates from chicken samples examined, with 100% agreement at the genus level to 94% at the species level as compared with conventional methods.     

动物源弯曲菌分离纯化鉴定方法优化

【目的】弯曲菌是重要的食源性人畜共患病原菌,通常难以诊断,而动物源方向缺少系统的检测分析方法及相应的优化。为了获得更适宜分离动物来源的空肠弯曲菌和结肠弯曲菌,提高分离效率,降低分离成本,特开展方法优化,填补动物源弯曲菌分离方法建设的空白,为耐药菌株的有效追踪溯源和风险评估提供依据。【方法】对已有的空肠弯曲菌和结肠弯曲菌分离纯化鉴定方法Preston肉汤和Bolton肉汤增菌;CCDA选择性培养分离、弯曲菌显色培养分离、Skirrow选择性培养分离;生化鉴定和分子生物学鉴定做了筛选优化,针对目前存在的分离纯化和鉴定方法进行了调查比较,得到了更适合于健康动物来源的粪便和盲肠中的分离方法。【结果】牛血清预增菌、CCDA选择性分离、弯曲菌显色培养基纯化、哥伦比亚血琼脂进行扩增及分子学方法鉴定,同时建议采用厌氧条件进行培养。该流程简单易行,成本较低,适合于绝大多数实验环境条件,是获取弯曲菌的首选流程,便于推广实施。【结论】通过将现研究阶段存在的大多数检测方法进行了综合分析,整理出一套针对于动物来源的粪便及肠道内容物的检测分析方法,对健康动物中弯曲菌的分离效率能够提高30%,同时空肠弯曲菌和结肠弯曲菌的分离效率无明显差异。     

Comparison of different sampling techniques and enumeration methods for the isolation and quantification of Campylobacter spp. in raw retail chicken legs

Comparable quantitative data of Campylobacter spp. on chicken products are a major data lack for quantitative risk assessment approaches. The objective of this study was to compare two different sampling techniques for the isolation and enumeration of Campylobacter spp. in chicken and to evaluate a suitable enumeration method comparing the most probable number (MPN) technique to the direct plating method. For this, 90 packages containing at least two raw chicken legs were examined for the comparison of sampling techniques, rinsing one leg and homogenizing 25 g of skin of the other leg of each package; both sample preparation types were examined by direct plating method and MPN technique in 40 out of 90 packages. Of the skin samples, 70% (63/90), and of the rinse samples, 77% (69/90), were Campylobacter-positive. Enumeration of Campylobacter spp. by direct plating revealed a median of log 4 cfu/leg surface in skin samples (S.D.=0.6) and a median of log 4.3 cfu/leg surface in rinse samples (S.D.=0.9) of the rinse samples; 73% (37/51) had higher numbers of Campylobacter spp. than the skin samples although the difference was not significant (p=0.08). The correlation coefficient of Campylobacter counts in skin and rinse samples was 0.43. The prevalence of Campylobacter spp. in rinse samples was 58% (23/40). In 5% (2/40) of the rinse samples, numbers of Campylobacter spp. could be detected only by the MPN technique due to the lower detection limit compared to the direct plating method. The MPN technique turned out to be unsuitable for the enumeration of Campylobacter spp. in skin samples because a layer formation on the top of the incubated MPN-tubes leads to irregular MPN results. Out of 80% (16/20) of the compared rinse samples, the direct plating detected higher numbers of Campylobacter spp., with a median count of log 4.2 cfu/leg surface (S.D.=1) compared the MPN technique where a median of log 4 cfu/leg surface (S.D.=1.1) was obtained. The difference was not significant (p=0.05). A highly positive correlation coefficient of 0.9 was observed between the direct plating and the MPN technique. Both sampling methods, rinsing the chicken leg and homogenizing the skin, are suitable for the detection and quantification of Campylobacter spp.; the direct plating method was superior to the MPN technique for enumerating Campylobacter spp. in raw chicken legs at retail level because enumeration is more rapid and less laborious.     

Detection of Campylobacter jejuni and Campylobacter coli from broiler chicken-related samples using BAX PCR and conventional International Organization for Standardization culture

In this study, the conventional International Organization for Standardization (ISO) culture method was compared with the DuPont Qualicon BAX system, a high-throughput, rapid molecular assay that can be used to detect several bacterial species, including Campylobacter jejuni and Campylobacter coli in diverse sample types. Standard enrichment culture is a time-consuming process, taking up to 6 days to obtain a confirmed result. Rapid molecular assays have been developed that provide results within 24 h. Naturally contaminated samples from the poultry production chain were examined for the presence of Campylobacter spp. Samples from broiler chicken ceca (n = 100), fresh chicken carcass rinses (n = 60), and bootsocks (gauze sock walked through a broiler chicken house; n = 50) were enriched according to the ISO 10272 method in Bolton broth specifically designed to detect Campylobacter spp. in complex sample types. Samples were enriched without blood for use with the BAX system using the Campylobacter BAX kits for the detection of C. jejuni and C. coli. Samples also were directly plated onto modified charcoal cefperazone deoxycholate agar, and results were compared with those from the enriched samples for the ability to detect Campylobacter spp. Campylobacter spp. were isolated from 49% of samples with conventional enrichment cultures, from 48% with direct culture, from 68% with the BAX system and enrichment cultures, and from 62% with the BAX system used directly with samples. Overall, the BAX system detected more positive samples than did the conventional culture method and is an effective methodology for the rapid and reliable detection of Campylobacter spp. from diverse sample types.     

Occurrence of Campylobacter spp. in raw and ready-to-eat foods and in a Canadian food service operation

The occurrence of Campylobacter spp. in a variety of foods from Ottawa, Ontario, Canada, and raw milk samples from across Canada was determined over a 2-year period. The samples consisted of 55 raw foods (chicken, pork, and beef), 126 raw milk samples from raw milk cheese manufacturers, and 135 ready-to-eat foods (meat products, salads, and raw milk cheeses). Campylobacter jejuni was detected in 4 of the 316 samples analyzed: 1 raw beef liver sample and 3 raw chicken samples. An isolation rate of 9.7% was observed among the raw chicken samples tested. This study also investigated the role of cross-contamination in disseminating Campylobacter from raw poultry within a food service operation specializing in poultry dishes. Accordingly, kitchen surfaces within a restaurant in Ottawa, Ontario, were sampled between March and August 2001. Tests of the sampling method indicated that as few as 100 Campylobacter cells could be detected if sampling was done within 45 min of inoculation; however, Campylobacter spp. were not detected in 125 swabs of surfaces within the kitchens of this food service operation. Despite the reported high prevalence of Campylobacter spp. in raw poultry, this organism was not detected on surfaces within a kitchen of a restaurant specializing in poultry dishes.     

Rapid and specific enzyme immunoassay on hydrophobic grid membrane filter for detection and enumeration of thermophilic Campylobacter spp. from milk and chicken rinses

Six commercially available anti-Campylobacter antibodies were examined for their applicability in an enzyme immunoassay on hydrophobic grid membrane filters, both for the detection and enumeration of Campylobacter spp. When a panel of nine Campylobacter (seven Campylobacter jejuni and two Campylobacter coli) and eight non-Campylobacter strains were used in a dot-blot format enzyme immunoassay to test the specificity of these antibodies, only one polyclonal antibody (Biodesign) detected all Campylobacter strains. Escherichia coli O157:H7 produced weak nonspecific signals due to endogenous peroxidase activity. The specificity of this Biodesign antibody was further tested against 30 more Campylobacter strains and more than 600 non-Campylobacter strains on hydrophobic grid membrane filters grown on modified Campylobacter agar with charcoal and deoxycholate, a Campylobacter selective medium. All the Campylobacter strains were detected, whereas only two (Acinetobacter calcoaceticus, Salmonella Minnesota) of the approximately 130 non-Campylobacter strains, which grew on modified Campylobacter agar with charcoal and deoxycholate, gave false-positive signals. This simple, rapid, and specific enzyme immunoassay also detected Campylobacter spp. from inoculated milk and chicken rinses and naturally contaminated chicken rinses.