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。     

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。     

环介导等温扩增法快速检测感染性腹泻病例标本中的空肠弯曲菌

目的 建立环介导等温扩增法(loop-mediated isothermal amplification, LAMP)快速检测感染性腹泻病例标本中的空肠弯曲菌。方法 收集2017年苏州市辖区3家医院的感染性腹泻患者的粪便样品326件, 用LAMP法对空肠弯曲菌的DNA进行检测, 在DNA扩增试剂盒提供的反应体系加入引物和模板进行空肠弯曲菌的LAMP反应, 测定LAMP方法检测空肠弯曲菌菌液的灵敏度; 同时用滤膜法进行空肠弯曲菌的分离培养, 参照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》分离鉴定空肠弯曲菌。结果 LAMP法检测空肠弯曲菌菌液的灵敏度是2.2 CFU/μL; 326件腹泻患者粪便中16件空肠弯曲菌LAMP检测为阳性, 阳性率为4.91%。共分离培养出14株空肠弯曲菌, 阳性率为4.29%。结论 针对感染性腹泻患者的粪便样本的空肠弯曲菌检测项目, 可以采用LAMP方法进行等温扩增初筛, 初筛阳性的样品再有针对性地进行后续的传统培养。     

空肠弯曲菌快速检测试剂盒研制及AOAC验证

本文自主研制出含内标的空肠弯曲菌实时荧光PCR快速检测试剂盒,并根据美国分析化学家协会(AOAC)微生物方法对试剂盒进行验证。结果表明,该试剂盒对115株目标菌和118株非目标菌的检测特异性为100%,灵敏度达6.4 CFU/mL。将试剂盒反复冻融30次,目的基因的Ct值无显著变化。稳健性测试结果显示,除了一株空肠弯曲菌对增菌液体积较为敏感外,增菌液体积、菌体裂解时间和DNA添加量均不影响其余10株空肠弯曲菌和5株同属的其它弯曲菌的检测结果。采用生产3 d、生产4个月和1年的3个批次试剂盒进行批间重复性测试和保质期测试,结果显示3批试剂盒对16株弯曲菌的检测结果一致,且11株空肠弯曲菌的Ct值无明显差异。对人工添加鸡肉、蔬菜和牛奶样品以及鸡肉实际样品检测结果显示,试剂盒方法与传统检测方法基本一致,未出现"假阴性"。试剂盒开放性好,可在不同品牌仪器上使用,含有的内部扩增对照可防止"假阴性",可应用于食品中空肠弯曲菌的快速检测。     

叠氮溴乙锭-环介导等温扩增法快速检测空肠弯曲菌活菌

目的建立叠氮溴乙锭(ethidium monoazide bromide, EMA)-环介导等温扩增(loop-mediated isothermal amplification,LAMP)快速检测空肠弯曲菌活菌的方法。方法以空肠弯曲菌mapA基因为检测靶基因,纯培养物提取模板进行LAMP反应,检测空肠弯曲菌LAMP灵敏度、EMA曝光照射时间试验和使用浓度试验。结果空肠弯曲菌LAMP检测灵敏度为800 CFU/mL;曝光照射时间为5 min; EMA终浓度50μg/mL以下时不会抑制空肠弯曲菌活菌的DNA扩增,终浓度为1μg/mLEMA能有效抑制4×10~4CFU/mL空肠弯曲菌死菌扩增; 1%活菌混合体系中EMA-LAMP检测结果为阳性。结论 EMA-LAMP方法能有效快速检测空肠弯曲菌活菌。     

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.     

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。     

食品中空肠弯曲菌荧光重组酶聚合酶扩增检测方法的建立

本研究为了实现空肠弯曲菌的快速和便捷检测,建立了一种基于荧光重组酶聚合酶扩增技术(exo-RPA)快速检测空肠弯曲菌的方法。通过对空肠弯曲菌和对照菌株的exo-RPA检测来判断该方法的特异性。用梯度稀释的空肠弯曲菌作为模板进行检测来分析exo-RPA方法的灵敏度。通过对模拟污染样品检测来分析exo-RPA的应用效果。分别以exo-RPA和荧光PCR检测实际食品样品来分析二者的检测效果。空肠弯曲菌exo-RPA方法可特异检出空肠弯曲菌,检测灵敏度达到6.0×102CFU/m L。在模拟污染试验中,含2.5×101 CFU/m L空肠弯曲菌的增菌液在培养24 h后可以被exo-RPA检测出阳性信号。exo-RPA和荧光PCR对于40份样品的检测结果相同。本研究建立的空肠弯曲菌exo-RPA具有特异、灵敏和抗干扰性强的特点,方法操作简便快速,具有较好的应用前景。     

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。     

辽宁省鸡源空肠弯曲菌耐药性监测与分析

目的了解辽宁省内鸡源空肠弯曲杆菌耐药性情况。方法采用特异性选择培养基分离出空肠弯曲菌,并使用生化反应和分子生物学方法双重方法进行鉴定,并进行耐药性分析。结果采集的辽宁省某屠宰场的鸡盲肠共198份样品,分离鉴定得到60株空肠弯曲菌,分离率为30.3%。同时对60株空肠弯曲菌进行了药物敏感性检测。结论 60株空肠弯曲菌几乎均对红霉素、庆大霉素、克林霉素和萘啶酸耐药,其MIC90值均高于所测试的最高药物浓度。空肠弯曲杆菌氟苯尼考和泰利霉素的敏感性最强。     

Epidemiology of Campylobacter enteritis

Campylobacter enteritis is the commonest form of infective diarrhoea in most developed countries of the world. In England and Wales laboratory reports indicate an annual incidence of about 85/100,000, but the true rate is probably nearer 1100/100,000. Measured costs run to many millions of pounds per year. Most infections are sporadic and believed to be foodborne; large outbreaks are infrequent and mostly due to the consumption of raw milk or unchlorinated water. Raw meats and animal products, notably broiler chickens, are the main source of campylobacters in food. A case-control study in the U.S.A., where eating habits are similar to those in Europe, attributed about one-half of human Campylobacter infections to the consumption of chickens. The production of Campylobacter-free chickens is not yet practicable, but considerable progress could be made to this end with sufficient motivation and resources from government and the poultry industry.     

Effects of short-chain nitrocompounds against Campylobacter jejuni and Campylobacter coli in vitro

ABSTRACT:  Effects of 2-nitro-1-propanol, 2-nitroethanol, nitroethane, and 2-nitro-methyl-propionate (0, 10, and 20 mM) on growth of Campylobacter jejuni were tested during culture in Bolton broth adjusted to pH 5.6, 7.0, or 8.2. The nitrocompounds were similarly tested against C. coli but at pH 8.2 only. Viable cell counts measured during incubation revealed main effects ( P < 0.05) of all nitrocompounds on the survivability of C. jejuni . An effect of pH ( P < 0.05) on the survivability of C. Jejuni during incubation with nitrocompounds was observed, with greater inhibition observed at pH 8.2 than at pH 5.6 or 7.0 for nitroethane, 2-nitro-l-propanol, and 2-nitroethanol, but not for 2-nitro-methyl-propionate, which showed greatest inhibition at pH 5.6. Except for 2-nitro-methyl-propionate, which was ineffective, all nitrocompounds elicited similar effects on C. coli . The effect of nitroethane and 2-nitro-l-propanol (10 mM) on naturally occurring Campylobacter was investigated during incubation of porcine fecal suspensions, where Campylobacter concentrations decreased more rapidly ( P < 0.05) in suspensions with added 2-nitro-l-propanol than in unsupplemented or nitroethane-supplemented suspensions, thus reiterating the superior inhibitory effect of 2-nitro-l-propanol.     

Serotypes and typability of Campylobacter jejuni and Campylobacter coli isolated from poultry products

Campylobacter infection is one of the most common bacterial enteric pathogens. Campylobacter jejuni and Campylobacter coli infections are mostly food- and waterborne and especially poultry is often assumed to be an important source. The heat-stable serotyping system (the 'Penner' scheme) was used to study the serotype distribution of C. jejuni and C. coli isolated from different food products of poultry origin sampled from retail outlets in Denmark. A total of 156 isolates were serotyped, 85% of these were C. jejuni and 15% were C. coli. The most common C. jejuni serotypes were O:2 (30%), O:1,44 (12%) and the O:4-complex (8%). O:46 was the most frequent serotype among C. coli isolates. These serotypes are also common among Danish clinical isolates and isolates from broiler chickens and cattle. Differences in serotype distribution were seen for different kinds of poultry products. Isolates from chicken products covered a large selection of serotypes. In contrast, the majority of the isolates from other product groups (turkey, poussin, wild birds) were concentrated on 1-3 serotypes. Using the standard procedure for antigen preparation and serotyping, 25 of the 156 strains (16%) were nontypable. This rate of nontypable isolates is significantly higher than experienced for isolates from other sources than food products, i.e faecal samples from animals and humans. Subculturing and re-typing of the nontypable isolates improved the typability. After two, five and 10 subcultures 16, six and one isolate became typable, respectively. Only three isolates (2%) remained nontypable after 10 subcultures.     

Antimicrobial resistance and molecular subtyping of Campylobacter jejuni and Campylobacter coli from retail meats

Campylobacter isolates (n = 297; 202 C. jejuni and 95 C. coli isolates) recovered from 2,513 retail meat samples (chicken breasts, ground turkey, ground beef, and pork chops) were examined for antimicrobial susceptibility. The isolates were further analyzed for genetic relatedness by pulsed-field gel electrophoresis (PFGE) using SmaI and KpnI restriction enzymes, and a subset of isolates (n = 174) were subtyped by multilocus sequence typing (MLST). The resistance most frequently observed was that to doxycycline (27.6%), followed by ciprofloxacin (13.8%) and erythromycin (6.4%). All isolates were susceptible to gentamicin and meropenem. C. coli showed higher resistance to doxycycline than did C. jejuni (42.1 versus 20.8%) and lower resistance to ciprofloxacin than did C. jejuni (10.5 versus 15.3%). Erythromycin resistance was only observed in C. coli. PFGE using SmaI plus KpnI digestion generated 168 clusters from 297 isolates: 115 from C. jejuni and 53 from C. coli. MLST revealed 44 sequence types (STs) under 10 clonal complexes from 120 C. jejuni and 27 STs under two clonal complexes from 54 C. coli. There was a positive association between PFGE and STs; however, PFGE showed greater discriminatory power than MLST. Subtyping data did not correlate with antimicrobial resistance phenotypes.     

Impediometric detection of Campylobacter coli

The rapid automated bacterial impedance technique (RABIT) was examined as a method for the detection of two wild-type isolates of Campylobacter coli in broth media. Both isolates failed to produce a change in impedance that was sufficient for detection in any combination of six nonselective basal broth media, including Mueller-Hinton broth, nutrient broth no. 2, brain heart infusion broth supplemented with yeast extract (0.5% [wt/vol]), brucella broth, Campy broth supplemented with yeast extract (0.5% [wt/voll), and Whitley impedance broth, at 37 and 42 degrees C. Although the strains did proliferate in the media, changes in conductivity were very small (ranging from 0 to 1,000 microS) and were not significantly greater than the drift in conductance observed in the control broth medium. Additional work is therefore required to define a nonionic growth substrate that will produce charged ions upon metabolism that are detectable by RABIT.     

Improved Culturing Techniques for Campylobacter

ABSTRACT Improved techniques for culturing and enumerating Campylobacter jejuni were developed. Parameters included (1) growth vessel type, (2) growth atmosphere, (3) incubation time, and (4) frequency of subcul-turing. Improved growth and consistent morphology were obtained using a tissue culture flask compared with an Erlenmeyer flask or a test tube as well as exposure to modified atmosphere (10% CO2, 85% N2, and 5% O2). When cultures of C. jejuni were incubated more than 24 h, transformation to the coccoid increased, and fewer colony-forming units were detected. Excessive subculturing (>2 times) resulted in increased formation of coc-coid forms. Decreased concentrations of colony-forming units of C. jejuni commonly attributed to the production of a viable, nonculturable form also may be due to cellular clumping. These techniques should alleviate difficulty and reduce variation when working with Campylobacter .     

弯曲菌及弯曲菌病的流行现状

为阐明弯曲菌在肠内外感染中的重要性 ,综述了弯曲菌病的发病率、流行特点、临床表现及弯曲菌对抗生素的耐药性。弯曲菌感染率在世界范围内呈普遍上升趋势 ,禽肉、水、牛奶、动物是主要传染源。空肠弯曲菌和结肠弯曲菌是弯曲菌感染最常见的两个种。格林巴利综合征是弯曲菌感染后最严重的并发症。近年来 ,弯曲菌对抗生素的耐药株迅速增多 ,最值得注意的是耐氟喹诺酮类弯曲菌。为更好地控制弯曲菌的感染与流行 ,各国 (特别是发展中国家 )有必要建立对弯曲菌的监测系统。