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...     

2种检测方法对弯曲菌检出率的影响

目的比较国标法和双孔滤膜法在生禽肉中弯曲菌的检测率,探索生禽肉中弯曲菌分离鉴定的有效检测方法。方法随机采集冷冻和新鲜禽肉产品共102份,通过国标法和双孔滤膜法2种方法检测,观察2种方法从样品处理、增菌和分离培养过程中的不同,并比较2种方法检测弯曲菌检出率的差异。结果102份样品中国标法检出弯曲菌的检出率为28.43%,其中空肠弯曲菌和结肠弯曲菌的检出率分别为13.725%和14.705%;双孔滤膜法检出弯曲菌的检出率为53.92%,其中空肠弯曲菌和结肠弯曲菌的检出率分别为26.47%和27.45%;双孔滤膜法检出率显著高于国标法,差异有统计学意义(P<0.05)。结论应用双孔滤膜法能显著提高生禽肉食品中弯曲菌的检出率。     

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。     

高分辨熔解曲线技术结合荧光实时PCR法同时检测鸡肉中4种食源性致病菌

建立高分辨熔解曲线技术同时快速检测鸡肉中沙门氏菌、单增李斯特菌、金黄色葡萄球菌和空肠弯曲菌。本研究分别以上述4种菌的fim Y、hly、nuc和orf C特异性基因为靶基因,建立能同时检测多种食源性致病菌的多重HRM-real time PCR检测体系,并对反应的特异性、灵敏度、重复性以及在人工染菌样品进行了评价。所建立的HRM-real time PCR检测体系对沙门氏菌、单增李斯特菌、金黄色葡萄球菌和空肠弯曲菌均产生特异性熔解曲线,Tm值分别为82.2℃、78.2℃、75.5℃和73.5℃;多重HRM-real time PCR反应体系检测单一致病菌菌液的灵敏度均为102拷贝数/m L,检测多种致病菌菌液的灵敏度为10~2拷贝数/m L;该反应体系重复性的试验内CV值在0.03%～0.32%之间,试验间CV值在0.45%～2.12%;人工染菌样本对4种菌的检测限均能达到5 CFU/25 g。结果表明该多重HRM-real time PCR检测体系可对上述4种禽肉中常见致病菌进行有效的检测与区分,特异性强,灵敏度高,反应重复性好,有望在食源性致病菌检测分析中发挥作用。     

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。     

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     

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。     

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。     

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。     

食品中单核细胞增生李斯特菌株的分离及聚合酶链式反应鉴定

目的 建立单核细胞增生李斯特菌的聚合酶链式反应(PCR)检测方法,了解市售食品中单核细胞增生李斯特菌的污染情况.方法 采集成都市市售生畜肉、生禽肉、熟肉制品、水产品、生食蔬菜以及其他熟食等食品样品共135份,采用李氏增菌肉汤(LB1,LB2)进行初增菌,应用选择性分离培养基PALCAM和在TSA-YE平板上进行分离,利用单增李斯特显色平板进行鉴定;根据李斯特菌的特异性基因iap基因设计引物,采用PCR方法检测所有分离的李斯特菌株;根据单增李斯特菌的特异性基因hly基因和prfA基因设计引物检测单核细胞增生李斯特菌株.结果 135份样品中共检出李斯特菌17株,检出率为12.6％;其中单核细胞增生李斯特菌4株,检出率为3.0％.结论 本研究建立的PCR方法具有特异性,本市市售食品不同程度受到李斯特菌的污染.     

Contamination of Campylobacter spp. from foodstuff

Prevalence of Campylobacter spp. in foodstuff. All campylobacter spp. were identified as thermophilic campylobacter jejuni. campylobacter jejuni were most commonly isolated from crude birds samples (37%), both cooled and frozen. Campylobacter jejuni were recovered in 57% meat and semifinished items samples with contamination levels from the cooled samples of 270+/-470 colony forming units (cfu) per 100 g, frozen samples of 238+/-346 per 100 g and in 351% offal samples, with a contamination level from the cooled samples 100+/-360 per 100 g.     

Pathogens on meat and infection in animals - Establishing a relationship using campylobacter and salmonella as examples

A high proportion of human campylobacter and salmonella infections is likely to originate from farm animals, usually directly from the consumption of contaminated meat or milk. Surveillance shows that campylobacter and salmonella genotypes are shared between human case isolates, farm animals and foods, although with the latter there can be marked differences between infection frequency in live animals and contamination rates in raw foods. This is supported by a variety of data from around the world, using a range of different methods. In this paper the evidence for farm animals being the reservoir of human salmonella and campylobacter infection is presented. However, a note of caution is sounded about the complex nature of zoonotic diseases caused by these two pathogens. Thus, many salmonellas and campylobacter types found routinely in food animals do not appear to cause human infections. Is this and artefact of the surveillance and/or microbiological methods used or are some strains of these bacteria genuinely non-pathogenic in man?     

Mercury residues in free-grazing cattle and domestic fowl form the artisanal gold mining area of Geita district,Tanzania

Environmental contamination with mercury from artisanal gold mines in Tanzania has been widely reported. People living around mining villages keep domestic animals which are allowed to feed freely in mercury-contaminated areas. This study investigated Hg accumulation in the liver and muscle tissue of cattle and domestic fowl reared in mining villages. Total mercury levels up to 436 and 820 µg/kg wet weight were found in liver samples taken from cattle and domestic fowl, respectively. Significantly higher mercury concentrations were found in liver samples collected at mining villages (p < 0.05) than those taken from the reference area. While mercury concentrations in liver samples exceeded the acceptable maximum concentrations for humans set in the Netherlands and Poland, the Hg concentrations in muscle were below the limits of most countries. It is recommended that the keeping of freely grazing cattle and domestic fowl in or around artisanal gold mines should be avoided.     

Detection of Turkey, Duck, and Guinea Fowl Egg in Hen Egg Products by Species-Specific PCR

A simplex polymerase chain reaction (PCR) has been applied for the specific detection of hen, duck, turkey, and guinea fowl in egg products using species-specific primers targeting the mitochondrial cytochrome b genes. The species-specific PCR yielded excellent results for identification of duck, turkey, and guinea fowl eggs in hen egg products, since the detection of 0.1% of each species was achieved, in liquid egg products as well as in powders. The proposed method is then a potentially reliable and suitable technique in routine food analysis for the research of fraudulent species mixture practices.     

Comparison of automated BAX PCR and standard culture methods for detection of Listeria monocytogenes in blue Crabmeat (Callinectus sapidus) and blue crab processing plants

This study compared the automated BAX PCR with the standard culture method (SCM) to detect Listeria monocytogenes in blue crab processing plants. Raw crabs, crabmeat, and environmental sponge samples were collected monthly from seven processing plants during the plant operating season, May through November 2006. For detection of L. monocytogenes in raw crabs and crabmeat, enrichment was performed in Listeria enrichment broth, whereas for environmental samples, demi-Fraser broth was used, and then plating on both Oxford agar and L. monocytogenes plating medium was done. Enriched samples were also analyzed by BAX PCR. A total of 960 samples were examined; 59 were positive by BAX PCR and 43 by SCM. Overall, there was no significant difference (P ≤ 0.05) between the methods for detecting the presence of L. monocytogenes in samples collected from crab processing plants. Twenty-two and 18 raw crab samples were positive for L. monocytogenes by SCM and BAX PCR, respectively. Twenty and 32 environmental samples were positive for L. monocytogenes by SCM and BAX PCR, respectively, whereas only one and nine finished products were positive. The sensitivities of BAX PCR for detecting L. monocytogenes in raw crabs, crabmeat, and environmental samples were 59.1, 100, and 60%, respectively. The results of this study indicate that BAX PCR is as sensitive as SCM for detecting L. monocytogenes in crabmeat, but more sensitive than SCM for detecting this bacterium in raw crabs and environmental samples.     

Prevalence and risk factors associated with campylobacter infections in broiler flocks in Shiraz, southern Iran

Campylobacter species are among the most common bacterial causes of human gastroenteritis in many countries, and poultry meat is considered as a major source of human campylobacteriosis. The present study was conducted to determine the prevalence of infection by Campylobacter jejuni and Campylobacter coli in broiler flocks in Shiraz and to investigate the possible risk factors for the campylobacter infections in this area. For detection of campylobacter, multiplex polymerase chain reaction (mPCR) was used. Between August and September 2009, a total of 100 broiler flocks from 100 commercial broiler farms were selected at slaughter and campylobacter status was determined by mPCR on caecal samples. Data about farms and flocks were collected by questionnaires. Approximately 76% (95% CI: 67-84%) of the flocks were positive for C. jejuni or C. coli. Twenty two percent were positive for C. jejuni, 32% for C. coli and 22% for both species. Results of the statistical analysis using multivariable logistic regression showed that the odds of flock infection decreased when level of owner's education (years) increased (OR = 0.86, P = 0.04), also odds of infection was nearly five times higher when age at slaughter was ≥ 45 days compared with < 45 days (OR = 5.3, P = 0.003) and use of antibiotic medications at early stage of production period was negatively associated with the infection status of the flock (OR = 0.33, OR = 0.059). We found no evidence of the effects of any other factors such as time interval between successive flocks, hygiene measures and number of broiler houses on the farm on the prevalence of campylobacter infection. Getting more attention to the health education issues and planning qualitative studies to reveal the behavioral aspects of the management policy, may be subjects of interest for future researches.     

Diversity of flaA genotypes among Campylobacter jejuni isolated from six niche-market poultry species at farm and processing

PCR-restriction fragment length polymorphism of the flagellin (flaA) gene in Campylobacter jejuni was used to determine the relationships of isolates collected at the farm and throughout processing for six niche-market poultry species. This study focused on two specialty chicken products, poussin and free range, and four other specialty products, squab, duck, guinea fowl, and quail. Cloacal and carcass samples were collected from three flocks from each of the six niche species. Three processing plants in California participated in a 2-year investigation. A total of 773 isolates from farm, posttransport, and the processing plants were genotyped, yielding a total of 72 distinct flaA profiles for the six commodities. Genetic diversity of C. jejuni at the farm was greatest for ducks with up to 12 distinct flaA types in two flocks and least for squab 1 flaA type between two farms. For two of the guinea fowl flocks, one free-range flock, two squab flocks, and all three poussin flocks, the flaA types recovered at the prepackage station matched those from the farm. Cross-contamination of poultry carcasses was supported by the observation of flaA types during processing that were not present at the farm level. New C. jejuni strains were detected after transport in ducks, guinea fowl, and free-range chickens. Postpicker, postevisceration, and prewash sampling points in the processing plant yield novel isolates. Duck and free-range chickens were the only species for which strains recovered within the processing plant were also found on the final product. Isolates recovered from squab had 56 to 93% similarity based on the flaA types defined by PCR-restriction fragment length polymorphism profiles. The 26 duck isolates had genetic similarities that ranged from 20 to 90%. Guinea fowl and free-range chickens each had 40 to 65% similarity between isolates. Poussin isolates were 33 to 55% similar to each other, and quail isolates were 46 to 100% similar. Our results continue to emphasize the need to clean processing equipment and posttransport crates in order to decrease cross contamination between flocks. This study also determined that several strains of C. jejuni had unique flaA types that could only be recovered in their host species.     

泰和乌骨鸡鸡肉总磷脂含量及其侧链脂肪酸组成的特性

本文比较了相同条件养殖的泰和乌骨鸡和两种非药用鸡鸡肉的总磷脂含量,并以GC-MS联用技术对三种鸡肉中磷脂的疏水侧链脂肪酸组成进行了鉴定和分析,用面积归一法,计算各脂肪酸的相对百分含量。结果表明,泰和乌骨鸡鸡肉中总磷脂含量显著高于非药用鸡,而两种非药用鸡间无显著差异;泰和乌骨鸡鸡肉磷脂侧链脂肪酸中多不饱和脂肪酸和花生四烯酸含量显著高于非药用鸡。     

云南盐津乌骨鸡与武定鸡肌肉蛋白质组学差异研究

选取云南两种具有代表性的地方优质鸡种盐津乌骨鸡和武定鸡为研究对象,采用双向电泳(2DE)分析其腿肌与胸肌中蛋白表达差异,并以基质辅助激光解析电离-串联飞行时间质谱(MALDI-TOF/TOF MS)鉴定腿肌与胸肌中差异蛋白质点。结果表明,经双向电泳图谱分析所获得的肌肉组织蛋白点分布于pH4.0~7.0之间,其中盐津乌骨鸡腿肌、胸肌中分别检测到(1421±34)、(1329±25)个蛋白点;武定鸡腿肌、胸肌中检测到(1585±19)、(1516±28)个蛋白质点。对比分析盐津乌骨鸡与武定鸡的DE图谱,发现有178个点存在明显的表达差异,在盐津乌骨鸡中高表达的点有72个,在武定鸡中高表达的点有106个。选取的蛋白质点经鉴定主要为核纤层蛋白A、磷酸葡萄糖变位酶1、β-烯醇酶、原肌球蛋白1、三磷酸腺苷合成酶、C-反应蛋白前体、B链α-晶状体蛋白、肌酸激酶M型、乙二醛酶1、磷酸甘油酸激酶 PGK1、慢骨肌肌钙蛋白T、蛋白酶体非ATP酶调节亚单位14、热休克蛋白β-1超氧化物歧化酶、以及一些假设蛋白和非特征性蛋白。     

Using a portable real-time PCR assay to detect Salmonella in raw milk

The purpose of this study was to determine the efficacy of a portable real-time PCR system in detecting Salmonella spp. in raw milk. The 200 bulk milk samples chosen for this study constituted a subset of the samples for a larger study; this subset contained 24 samples that were culture positive for Salmonella and 176 that were culture negative. Milk was both plated directly on selective agar and plated after enrichment in selective media. Presumptive Salmonella colonies were isolated by direct culturing of five samples, while Salmonella was isolated from the remaining 19 positive samples only after enrichment. Presumptive Salmonella isolates were serotyped, and isolates from 22 samples were confirmed to be Salmonella isolates. PCR assays of culture-positive milk prior to enrichment yielded no evidence of Salmonella. DNA extracts of bacterial pellets from the enriched samples were analyzed for Salmonella by real-time PCR with the Ruggedized Advanced Pathogen Identification Device (RAPID). Fifty-four samples from the enrichment pellets tested positive for Salmonella by real-time PCR. Two samples that tested positive for Salmonella by culture and serotyping tested Salmonella negative by real-time PCR. Serotyping identified isolates from these samples as Salmonella Montevideo. All DNA extracts of Salmonella Montevideo isolates tested positive for Salmonella by real-time PCR. Thirty-three samples tested negative by culture and positive by real-time PCR. These results indicate that the portable real-time PCR system appears to be a useful tool for detecting Salmonella in raw milk. Additionally, the combination of enrichment and real-time PCR techniques used in this study can yield results in 24 h, compared with the 48 to 72 h required for traditional culture.