多重PCR鉴定动物源空肠弯曲菌和结肠弯曲菌方法的建立

建立鉴定空肠弯曲菌和结肠弯曲菌的多重PCR(mPCR)方法。方法 分别以16S rRNA、马尿酸酶和16S-23S rRNA基因为靶序列设计特异性引物,建立多重PCR方法检测37株菌株样品,同时采用ⁱⁿgene CAM nested PCR检测试剂盒检测验证,进行结果比较分析。结果 该多重PCR方法可扩增出空肠弯曲菌和结肠弯曲菌的特异性条带,其他对照菌株均未扩增出条带,具有较好的特异性;检测敏感性可达0.81pg/μl空肠弯曲菌DNA,0.93pg/μl结肠弯曲菌DNA。多重PCR方法和试剂盒检测结果的符合率为100%,二者与国标GB/T 4789.9—2008方法的符合率达97%以上。结论 本试验建立的多重PCR方法操作快速方便、节约试验成本,具有较好的特异性、敏感性和重复性,可用于弯曲菌的鉴定。     

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。     

肉鸡屠宰加工过程中空肠弯曲菌污染的监测与分析

目的 调查分析浙江金华地区肉鸡屠宰加工过程中空肠弯曲菌的污染现状及规律。方法 选择金华地区6家肉鸡屠宰加工企业, 通过直接计数法对空肠弯曲菌定性定量检测, 分析其流行病学规律。结果 采集的2139份样品中, 泄殖腔、脱毛、取内脏、消毒预冷、包装和速冻等环节样品的空肠弯曲菌阳性率分别为92.48%、83.39%、98.12%、79.31%、86.21%和77.45%, 空肠弯曲菌阳性数分别为12926.14±1821.84 CFU/g、379.01±67.44、856.66±206.27、110.57±19.52、138.01±76.5、67.90±46.79 CFU/100 cm2。总体上, 空肠弯曲菌阳性率和阳性数均呈现出先升高、后降低、再升高的变化规律, 大型企业的空肠弯曲菌污染情况较中小型企业要好。结论 所检测的6家肉鸡屠宰加工企业均被空肠弯曲菌污染, 大型企业应重视包装环节的生产工艺和卫生消毒, 中小型企业更要严格把控包装环节的操作规范, 提高脱毛和消毒预冷工艺。     

辽宁省市售生禽肉食品中空肠弯曲菌毒力基因的鉴定

目的 通过对辽宁省生禽肉中空肠弯曲菌的污染状况进行调查,了解其生禽肉中空肠弯曲菌的毒力基因携带特点.方法 按照GB 4789.9—2014《食品安全国家标准食品微生物学检验空肠弯曲菌检验》及采用PCR扩增技术和哥伦比亚血平板检测的方法,对2018—2020年采自辽宁省14个监测点收集的335份生禽肉进行空肠弯曲菌的检测...     

空肠弯曲菌与食品

近几年来空肠弯曲菌(Campylahacterjej－uni)已被世界各国公认为是引起人类急性腹泻,特别是婴儿腹泻的重要病原菌。空肠弯曲菌可以多种方式从动物宿主传播给人,也可通过直接接触污染的动物宰体传播。但是,更多的是通过摄入污染的食物或水而间接传播的,...     

徐州市禽源弯曲菌的分离鉴定及耐药性分析

目的 了解徐州市市售禽肉中弯曲菌的污染状况及对抗生素的耐药情况。方法 随机采集农贸市场及超市新鲜或冷冻禽肉72份,采用滤膜法分离培养鉴定弯曲菌;采用琼脂稀释法测定分离出的菌株对6类11种抗生素的耐药性。结果 72份样品中共检出29份弯曲菌,总检出率为40.28%（29/72）,空肠弯曲菌和结肠弯曲菌检出率分别为19.44%（14/72）和20.83%（15/72）;新鲜禽肉和冷冻禽肉检出率分别为68.72%（22/32）和17.5%（7/40）,差异有统计学意义（χ2=19.412,P<0.05）,空肠弯曲菌在新鲜禽肉和冷冻禽肉中检出率分别为34.38%（11/32）和7.5%（3/40）差异有统计学意义（χ2=8.198,P<0.05）,结肠弯曲菌在新鲜禽肉和冷冻禽肉中检出率分别为34.38%（11/32）和10%（4/40）差异有统计学意义（χ2=6.404,P<0.05）;空肠弯曲菌耐药较高的五种抗生素为：萘啶酸100.00%(14/14)、环丙沙星100.00%(14/14)、四环素100.00%(14/14)、 氟苯尼考64.29%(9/14) ,庆大霉素28.57%(4/12);结肠弯曲菌耐药较高的五种抗生素为：萘啶酸100.00%(15/15)、环丙沙星100.00%(15/15)、四环素100.00%(15/15)、链霉素86.67%(13/15),庆大霉素80%(12/15) ;两种弯曲菌的多重耐药性均为100.00%。结论 徐州市市售禽肉中弯曲菌污染率较高,其中新鲜禽肉较冷冻禽肉中检出率高,徐州地区弯曲菌耐药情况严重     

Fla-DGGE法对食品中空肠弯曲菌和结肠弯曲菌的检测和分型

本文应用鞭毛蛋白基因flaA和flaB的变性梯度凝胶电脉(denaturing gradient gel electrophoresis,DGGE)方法对食品中空肠弯曲菌和结肠弯曲菌进行检测和分型。采用RBB(rpeated bead beating)、CTAB和丙酮-氯仿抽提三种方法提取样品基因组后进行fla-DGGE检测,结果完全一致。10份生鲜鸡、鸭肉样品中有8份含有空肠弯曲菌或结肠弯曲菌,其中3份含有两种或两种以上的空肠弯曲菌或结肠弯曲菌或它们的不同型别。克隆测序后结果表明,有7份样品被同一种空肠弯曲菌所污染,其中3份样品还被同一种结肠弯曲菌所污染,1份样品被污染情况严重,检测到含有一种结肠弯曲菌和三种空肠弯曲菌。Fla-DGGE方法快速、准确、灵敏度高,可用于食品中空肠弯曲菌和结肠弯曲菌的快速检测和分型。     

空肠弯曲菌致病分子机制研究进展

空肠弯曲菌(Campylobacter jejuni)是人类的食源性病原菌,感染后主要引起急性肠炎,与人类格林-巴利综合症也有密切关系.研究表明,空肠弯曲菌致病性是多种毒力因子共同作用的结果.作者从分子生物学研究水平综述空肠弯曲菌黏附、定植、侵入、产细胞毒素、分子模拟等致病机制.     

双重PCR 方法检测沙门氏菌和空肠弯曲菌

为建立能够同时检测食品中沙门氏菌和空肠弯曲菌的双重PCR 方法。采用沙门氏菌鞭毛基因fimY 和空肠弯曲菌马尿酸酶基因hipO 设计特异性引物,并对影响PCR 扩增的主要因素——引物浓度、退火温度、Mg2+ 浓度因素进行优化,比较单一PCR 和双重PCR 的检测效果。结果表明：采用单一PCR 法检测沙门氏菌和空肠弯曲菌时,灵敏度分别可达到3.98pg 和4.05pg;而采用双重PCR 检测时,灵敏度较单一PCR 法有所下降,沙门氏菌和空肠弯曲菌检出限量分别为398pg 和40.5pg。本研究建立的特异性强和灵敏度高的双重PCR 检测方法,可为实现食品中沙门氏菌和空肠弯曲菌的同时检测提供新方法。     

PCR技术检测空肠弯曲菌的研究进展

空肠弯曲菌(Campylobacter jejuni)是一种人畜共患病病原菌,可以使人和动物引发多种疾病。目前,检测C.jejuni采用的国标方法是传统的培养法,但C.jejuni培养条件苛刻,且培养法存在操作繁琐、特异性不强、费时等缺点。聚合酶链式反应(polymerase chain reaction,PCR)以其快速、准确、灵敏度高、特异性强的特点,现已广泛应用于C.jejuni的检测,并成为目前快速检测临床与食品中C.jejuni最常用的的方法。本文综述了近年来利用RCR技术,包括常规PCR、多重PCR、巢式PCR、实时荧光定量PCR、PCR-酶联免疫吸附法、最大几率数-PCR、PCR-限制性片段长度多态性、PCR-变性高效液相色谱、PCR-变性梯度凝胶电泳和磁捕获-荧光PCR方法检测C.jejuni的研究进展,并针对这些PCR技术的原理、检测效果、优点和缺点等方面进行了分析比较,为有效控制和预防该菌引起的疾病提供重要信息。     

东北市售鸡肉中空肠弯曲杆菌的分离鉴定、分型及耐药性分析

为了揭示东北地区市售鸡肉中空肠弯曲杆菌的污染情况、种群特征及耐药性。采集东北地区市售鸡肉样品1 000份,分离鉴定空肠弯曲杆菌。通过多位点序列分型技术分析空肠弯曲杆菌的遗传多样性。利用K-B琼脂扩散法检测该致病菌对8种抗生素的耐药性。结果表明:1 000份样品中共分离出62株空肠弯曲杆菌,污染率为6.2%;62株空肠弯曲杆菌被分为14个序列型(Sequence Type,ST),其中ST5和ST1为该种群的优势ST;耐药性分析结果显示,62株空肠弯曲菌对环丙沙星、复方新诺明和头孢噻肟具有高度的敏感性。     

Effect of refrigerated and frozen storage on the survival of Campylobacter jejuni in cooked chicken meat breast

This experimental work aimed to examine the survivability of Campylobacter jejuni in cooked chicken breast under several conditions: storage for 1, 3, and 7 d at refrigerated temperatures (4 °C) and for 20 d at frozen temperatures (-18 °C). In addition, storage at ambient temperature (26 to 28 °C) was involved. Chicken samples were inoculated with a mixed culture of C. jejuni strains (ATCC: 29428 and 33219) of known concentrations (50 and 500 CFU/g). Bacterial cells were recovered and enumerated using standard procedure (Preston method). Bacteria were not detected in the majority of samples stored at ambient temperature. Refrigeration reduced survivals in 95, 90, and 77.5% for samples inoculated with 500 CFU/g and kept for 1, 3, and 7 d, respectively. The maximum reduction reached 1 log(10) cycle for all refrigeration durations. It was observed that bacteria died in 17.5% of samples kept for 7 d at 4 °C. However, survivors in samples inoculated with 50 CFU/g were not detected in 50, 65, and 55% of samples kept for 1, 3, and 7 d, respectively. Freezing rendered survivors not detectable in 70% of samples inoculated with 50 CFU/g, while survived viable counts were reduced in 92.5% of samples inoculated with 500 CFU/g. These findings suggested that C. jejuni could be killed or just sublethally injured with or without reduction in viable counts under the investigated storage temperatures, which may indicate the ability of this bacterium to survive in chicken meat stored under refrigerated and frozen conditions.     

Validation of an Improved Method for Detection of Campylobacter jejuni in Foods

ABSTRACT:  Campylobacter jejuni ATCC 29428 and 33560 were inoculated separately into beef muscle, ground beef, and chicken skin to yield approximately 10 to 100 CFU/g of food sample. The samples were stored at 4 °C for 10 d. On days 0, 3, 7, and 10, enrichment cultures in Bolton broth supplemented with antibiotics, with and without blood supplementation were made for each sample, for 24 and 48 h following the Food and Agricultural Products Center (FAPC) and the Food and Drug Administration (FDA) protocols. Enumeration of the organisms in the enrichment cultures was done on Campylobacter Karmali selective agar after 24 and 48 h of enrichment to compare the extent of growth in both protocols. There were no significant differences between counts recovered using the FDA and the FAPC methods for detection of Campylobacter jejuni for either strain in any of the food products tested ( P > 0.05). No significant differences were observed in performance of enrichment broth supplemented with and without blood ( P > 0.05). After 48 h of enrichment, the counts recovered were similar for all products. The organisms were detectable on all days of storage in raw chicken skin, beef, and ground beef samples after both 24 and 48 h of enrichment. The results from the FAPC method for detection of C. jejuni from food were not different from the FDA method. While in the proposed method incubation at 37 °C was adequate for the strains tested it is recommended that both enrichment temperatures be used for naturally contaminated samples to ensure detection of all strains that might be present.     

新疆库尔勒市鸡肉源沙门氏菌的分离鉴定及 耐药性分析

目的了解新疆库尔勒市鸡肉沙门氏菌污染状况和沙门氏菌分离株对常用抗菌药物的耐药性。方法参考食品安全国家标准GB 4789.4-2010,利用生化反应与普通PCR方法结合进行沙门氏菌分离鉴定,采用微量肉汤稀释法进行常见抗菌药物敏感试验。结果 180份鸡肉样品共检出沙门氏菌阳性样品27份,分离鉴定到阳性沙门氏菌28株,污染率为15.00%;10种抗生素药物敏感试验表明:沙门氏菌分离菌株对甲氧苄啶的耐药率高达100%,氯霉素为89.30%、磺胺异噁唑为85.70%,四环素为78.60%、氨苄西林为71.40%、阿莫西林为71.40%、链霉素为64.30%、环丙沙星为53.60%,对诺氟沙星和氟苯尼考的耐药率低于50%,分别为32.10%和25.00%。结论新疆库尔勒市鸡肉中沙门氏菌存在一定程度的污染,耐药状况比较严重。     

空肠和结肠弯曲菌鞭毛蛋白基因fla A的检测

目的 运用一种快速、敏感、特异的检测空肠和结肠弯曲菌的方法.方法 以空肠和结肠弯曲菌所共有特异的鞭毛蛋白基因 fla A的一段高度保守序列为引物,用PCR法扩增fla A基因上的一段约1 700 bp的片断.用该引物对空肠和结肠弯曲菌的标准株、福建省的食品分离株进行PER扩增检测,并同时检测该PCR方法的敏感性.结果 扩增片断表现出极好的特异性,2株空肠和结肠弯曲菌标准菌株、8株分离自不同食品样品的空肠穹曲菌和结肠弯曲菌菌株均为阳性,且敏感性实验显示该PCR方法的反应体系最低检出菌量为6 CFU.结论 该方法快速、敏感、特异,可用于突发性食物中毒和暴发感染的调查.     

Antimicrobial edible apple films inactivate antibiotic resistant and susceptible Campylobacter jejuni strains on chicken breast

Abstract: Campylobacter jejuni is the leading cause of bacterial diarrheal illness worldwide. Many strains are now becoming multidrug resistant. Apple‐based edible films containing carvacrol and cinnamaldehyde were evaluated for bactericidal activity against antibiotic resistant and susceptible C. jejuni strains on chicken. Retail chicken breast samples inoculated with D28a and H2a (resistant strains) and A24a (a sensitive strain) were wrapped in apple films containing cinnamaldehyde or carvacrol at 0.5%, 1.5%, and 3% concentrations, and then incubated at 4 or 23 °C for 72 h. Immediately after wrapping and at 72 h, samples were plated for enumeration of viable C. jejuni. The antimicrobial films exhibited dose‐ and temperature‐dependent bactericidal activity against all strains. Films with ≥1.5% cinnamaldehyde reduced populations of all strains to below detection at 23 °C at 72 h. At 4 °C with cinnamaldehyde, reductions were variable for all strains, ranging from 0.2 to 2.5 logs and 1.8 to 6.0 logs at 1.5% and 3.0%, respectively. Films with 3% carvacrol reduced populations of A24a and H2a to below detection, and D28a by 2.4 logs at 23 °C and 72 h. A 0.5‐log reduction was observed for both A24a and D28a, and 0.9 logs for H2a at 4 °C at 3% carvacrol. Reductions ranged from 1.1 to 1.9 logs and 0.4 to 1.2 logs with 1.5% and 0.5% carvacrol at 23 °C, respectively. The films with cinnamaldehyde were more effective than carvacrol films. Reductions at 23 °C were greater than those at 4 °C. Our results showed that antimicrobial apple films have the potential to reduce C. jejuni on chicken and therefore, the risk of campylobacteriosis. Possible mechanisms of antimicrobial effects are discussed. Practical Application: Apple antimicrobial films could potentially be used in retail food packaging to reduce C. jejuni commonly present on food.     

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。