PCR法检测动物源性食品中布氏弓形菌

弓形菌是一种新型食源性致病菌,其中以布氏弓形菌污染率最高。本研究采用扩增23S rRNA的PCR法特异性检测布氏弓形菌,方法灵敏度可达103 CFU/mL。2株布氏弓形菌均特异性地扩增出了长度为2061 bp的条带;嗜低温弓形菌、斯氏弓形菌、空肠弯曲菌等共18株不同种类的菌株均无扩增产物出现,表明此PCR法能特异性的将布氏弓形菌鉴定到种一级水平。比较实验结果表明,API CAMPY鉴定试剂盒对于布氏弓形菌鉴定仅能到弓形菌属一级水平,本PCR法能实现布氏弓形菌种一级水平的鉴定,用于布氏弓形菌的检测具有优势。55份动物源性食品样品用Johnson-Murano肉汤增菌后用PCR法进行检测,其中5份样品为布氏弓形菌阳性,阳性检出率为9.1%。上述实验结果表明,本方法特异性强、操作简便,节省了检测时间,可用于动物源性食品中布氏弓形菌的快速检测。     

食品中弓形菌16S rRNA特异性扩增检测方法的建立

针对弓形菌16SrRNA基因合成1对引物,通过对聚合酶链式反应(PCR)扩增条件的优化,建立了检测弓形菌的PCR方法。3株弓形菌标准菌株PCR产物测序结果与NCBI上公布的弓形菌16S rRNA基因序列进行比对,比对结果表明3株弓形菌测序结果与NCBI上公布的弓形菌16S rRNA基因序列同源性均在99%以上。3株弓形菌标准菌株均特异性地扩增出了长度为1202bp的片段,其他19株不同种类的菌株均无扩增产物出现。55份食品样品用Johnson-Murano肉汤增菌后用此法进行检测,其中6份样品为弓形菌阳性,阳性率为10.9%。上述实验结果表明,方法特异性强、操作简便,节省了检测时间,可用于食品中弓形菌的快速检测。     

应用gyrA基因聚合酶链反应检测食品中弓形菌

建立了一种快速、敏感、特异检测弓形菌的PCR方法。选取弓形菌gyrA基因序列作为靶序列设计特异性引物Arco/gyrAF和Arco/gyrAR,进行PCR扩增实验。结果弓形菌呈现出一条298 bp的特异性条带,其余非弓形菌均无阳性扩增。灵敏度实验结果表明,该法可以检测到82.6 pg的DNA。经过对16份生鲜鸡肉样品的检测,9份样品中检测到弓形菌,样品阳性率为56.25%。该PCR方法可以高效、灵敏地检测食品中弓形菌,具有很强的实际应用价值。     

Taqman探针荧光PCR法检测食品中的大肠埃希氏菌O145

针对大肠埃希氏菌O145的O抗原基因簇的wck D基因的特异性序列设计引物和Taqman探针,建立检测大肠埃希氏菌O145的荧光PCR方法,对其灵敏度、特异性进行验证,并将其用于食品样品的检测。结果表明,本研究中的方法可实现对大肠埃希氏菌O145的特异性扩增,其它27株非O145大肠埃希氏菌和20株非大肠埃希氏菌细菌的菌株均无扩增;检测的灵敏度可达165拷贝/反应;339份食品样品EC肉汤增菌后用本荧光PCR法进行检测,检出大肠埃希氏菌O145阳性31份,阳性率为9.1%。实验结果表明,本研究成功建立了可用于食品中大肠埃希氏菌O145的Taqman探针荧光PCR方法,食品样品采用EC肉汤增菌24 h、热裂解法提取核酸,增菌后检测所需时间由至少3 d~7 d缩短为仅需2 h~3 h,食品检测全过程仅需约28 h,经证实本方法特异性强、操作简便,为食品中大肠埃希氏菌O145提供了一种的快速检测手段。     

PCR技术检测动物源性食品中沙门菌的应用研究

为快速准确检测动物源性食品中的沙门菌,将建立并优化的沙门菌PCR检测试剂盒应用于动物源性食品的检测,并于国标法进行了比较.对上海地区238份鸡蛋、685份原料牛奶、283份猪肉、314份牛肉和58份虾仁样品的沙门菌检测表明,PCR法的敏感性和特异性均为100%,与国标法的符合率亦为100%,且检测时间仅为2 d,较国标法大为缩短.该方法可用于动物源性食品中沙门菌的检测,并具有快速、特异、敏感等优点。     

石家庄市零售鸡肉样品中弓形杆菌污染调查

目的调查石家庄市零售鸡肉中弓形杆菌的污染情况,为石家庄市弓形杆菌分布特征和防控提供基础数据支持。方法从石家庄市市内四区的菜市场、超市、连锁肉店随机购买零售鸡肉样品90份,应用驱动增强动力双孔滤膜法从样品中分离培养弓形杆菌,利用弓形杆菌多重聚合酶链式反应(PCR)检测及测序的方法对分离菌株进行种属鉴定,利用16S rDNA、rpoB基因测序的方法验证菌种鉴定结果。结果 90份鸡肉样品中,60份样品检出弓形杆菌86株,其中嗜低温弓形杆菌49株、布氏弓形杆菌37株,弓形杆菌检出率为66.67%(60/90)。60份阳性样品中,嗜低温弓形杆菌阳性样品45份(75.00%),布氏弓形杆菌阳性样品35份(58.33%)。嗜低温弓形杆菌和布氏弓形杆菌混合感染阳性样品20份,占比为22.22%(20/90)。不同采样点弓形杆菌检出率有较大差异。结论石家庄市零售鸡肉中弓形杆菌污染较重,嗜低温弓形杆菌污染率高于布氏弓形杆菌,两种弓形杆菌混合污染样品率较高。     

食品中单核细胞增生性李斯特氏菌PCR快速检测

通过扩增hly基因建立检测单核细胞增生性李斯特氏菌(Lm)的PCR方法.该方法具有较强的特异性,35株经传统方法鉴定的菌株PCR结果均为阳性,而其他三种同属异种菌,包括英诺克李斯特氏菌、绵羊李斯特氏菌和威尔斯李斯特氏菌及非李斯特氏菌均未扩增出特异性的片段.PCR方法对上Lm纯培养物的最低检测限为7.3CFU/μl,对模拟污染的生猪肉和蔬菜的检测低限为4CFU/g,牛奶为4CFU/ml.应用该方法对285份食品样品检测,17份样品Lm呈阳性,结果与常规的分离培养方法完全一致.该种方法具有敏感、特异、快速及准确的优点,可用于食品中Lm的快速检测.     

环介导等温扩增法检测动植物源性单核细胞增生李斯特氏菌

目的采用环介导等温扩增技术检测动植物源性单核细胞增生李斯特氏菌。方法在北京市范围内各业态餐厅购买600份凉菜样品为研究对象,以单核细胞增生李斯特氏菌的hlyA基因为目标基因,设计4条特异性引物,使用环比等温扩增法(loop-to-isothermal amplification, LAMP)创建凉菜中单核细胞增生李斯特氏菌快速检测方法并将其应用于鉴定凉菜中分离的单核细胞增生李斯特氏菌。结果通过细菌分离培养法对600份凉菜进行检测,一共检测分离出37株致病菌菌株,其中包括7株单核细胞增生李斯特氏菌、10株沙门氏菌和20株大肠埃希氏菌,单核细胞增生李斯特氏菌的检出率为1.2%。用LAMP对样品中的菌株进行测定,7株凉菜中分离的单核细胞增生李斯特氏菌为阳性,其他菌株为阴性。通过LAMP方法检测动植物源性凉菜中分离的单核细胞增生李斯特氏菌平均检出限为2.2*10 CFU/μL、特异性为100%。动物源性凉菜中的致病菌含量较高。结论环介导等温扩增法具备灵敏、快速、操作简便的特点,适合用于餐饮行业的现场快速检测领域。     

实时荧光PCR法与环介导等温扩增法检测食品中动物源性成分的比较

目的比较实时荧光PCR(real-time PCR)法及环介导等温扩增(loop-mediated isothermal amplification,LAMP)检测食品中动物源性成分,为食品安全监管部门动物源性成分鉴定提供准确、高效的检测方法。方法采用实时荧光PCR法及LAMP法分别对市售肉及其制品进行牛、猪和鸡源性成分鉴定,并与标签明示的肉源成分比对,以准确性和时效性2个指标对上述2个方法进行评价。结果 Real-time PCR法检出4份样品与标签明示肉源不符,LAMP法检出5份样品与标签明示肉源不符,而16份样品中仅有1份样品2种方法检测结果不同。Real-time PCR法检测用时1.5 h,提取检测用DNA用时1.5 h,总用时3 h;而LAMP法检测用时45 min,提取检测用DNA用时20 min,总用时65 min。结论 LAMP法与Real-time PCR均具有较好的特异性、准确性,但LAMP法耗时短、成本低、操作简单,便于现场快速监督抽检。     

环介导等温扩增方法在食品中沙门菌检测的应用和评价

将环介导等温扩增检测方法应用于食品中沙门菌的检验,并在检测方法特异性、灵敏度等方面与实时荧光PCR和传统检测方法进行比较。方法 针对沙门菌属高度保守的fimY基因设计环介导等温扩增检测引物并优化反应体系,在特异性、灵敏度和实际样品检测等方面与实时荧光PCR及传统检测方法比对。结果 本研究建立的LAMP方法检测沙门菌93株和非目标菌31株,具有良好的特异性。在纯培养、无需增菌情况下,其检测灵敏度为6.4×10²cfu/ml,与实时荧光PCR方法相当。食品基质添加试验中,环介导等温扩增方法检测低限为2cfu/25g样品;对45份实际食品样品检测结果表明,该方法实际样品检出率为11.1%,与实时荧光PCR及传统方法检测结果一致。结论 本研究建立的沙门菌环介导等温扩增检测方法具有良好的特异性,检测灵敏度与实时荧光PCR相当,适用于沙门菌的快速筛选。     

Direct detection and identification of Arcobacter species by multiplex PCR in chicken and wastewater samples from Spain

Twenty-two chicken livers, 10 chicken carcasses, and 15 wastewater samples were processed and analyzed for Arcobacter by PCR and traditional culture methods. Samples were enriched for 24 and 48 h, incubated at 30 degrees C under aerobic conditions, and streaked on blood selective media. To determine the best isolation conditions, 20 samples also were processed under microaerophilic conditions at 37 degrees C. Simple and multiplex PCR assays were used directly with enrichment broths and isolated strains. Seventeen Arcobacter strains were isolated from chicken samples, and A. butzleri was the only Arcobacter species identified. The direct PCR assay revealed that 29 of the 32 chicken samples were contaminated with Arcobacter. A. butzleri was the most frequently detected species, although Arcobacter cryaerophilus also was present in some of the samples and Arcobacter skirrowii occasionally was detected. All the wastewater samples were positive by PCR assay for Arcobacter after 24 h of enrichment. A. butzleri and A. cryaerophilus were detected with the multiplex PCR assay. Fourteen Arcobacter strains were isolated from 10 of the 15 water samples analyzed; 7 were identified as A. butzleri and the remaining 7 were A. cryaerophilus. Both for chicken and water samples, Arcobacter detection rate for PCR amplification was higher than for culture isolation. These results indicate the high prevalence of Arcobacter in chicken and wastewater and the inadequacy of available cultural methods for its detection. The species-specific multiplex PCR assay is a rapid method for assessing Arcobacter contamination in chicken and wastewater samples and is a viable alternative to biochemical identification of isolated strains.     

One-step polymerase chain reaction-based typing of Arcobacter species

A species-specific PCR assay was developed for the identification of the Arcobacter species, Arcobacter butzleri, Arcobacter cryaerophilus 1A and 1B, and Arcobacter skirrowii. The primers, which amplify the most variable areas of the 23S rRNA gene, were designed to perform species-specific identification by one-step PCR. The DNA sequence of the region from A. cryaerophilus 1B was determined, and the specific pimer for the species was designed. By using one-step PCR containing the mixed primers N.butz, N.c1.A, N.c.1B, and N.ski, species-specific amplifications were detected from the reference strains of A. butzleri, A. cryaerophilus 1A, 1B, and A. skirrowii, respectively. Primers designed in this study were also evaluated on 10 of Japanese field isolates, and all species were identified. This simple one-step PCR assay was found to be a powerful tool for the survey of Arcobacter infection.     

Prevalence and diversity of Arcobacter spp. in the Czech Republic

The aim of this study was to examine 634 samples of chicken, lamb, pork, beef, fish, samples from the intensive animal industry and from poultry for slaughter, as well as from the domestic breeding of poultry, horses, pigs, and lambs, from surface water, and from clinical samples for the presence of Arcobacter. All the samples were examined with a cultivation method, followed by confirmation by multiplex PCR. The method of multiplex PCR applied directly to a liquid medium after enrichment was applied only to the samples with the highest probability of the presence of arcobacters. Arcobacter spp. were detected in 11.8% of the samples, of which A. butzleri, A. cryaerophilus, and A. skirrowii were found in 6.6, 5.1, and 0.2% of the samples, respectively. The sources of the arcobacters were chicken meat from the retail market, intensive animal production facilities, domestic chicken breeding facilities, lamb raising environments, surface water and wastewater, and beef swabs taken in a meat processing factory. No occurrence of arcobacters was identified in the swabs from slaughter turkeys, ducks, and wild poultry. No arcobacters were found in horse and pig breeding environments, on pork, or on the swabs of fish. Forty-two rectal swabs taken from humans were also free of Arcobacter. Seventeen isolates of Arcobacter were further identified by sequencing the 16S rRNA gene. Varied genotypes were observed among A. butzleri from chicken meat and chicken breeds, and A. cryaerophilus from wastewater and chicken breeds. They were similar to the genotypes present in wastewater, porcine feces, human stool, and human blood obtained from databases. Our results revealed that the chicken meat from the retail market is an important source of arcobacters. Cross-contamination during handling of chicken carcass practices could play a key role in the spread of Arcobacter.     

Sensitivity of three methods used in the isolation of Arcobacter spp. in raw ground pork

Arcobacter, an aerotolerant Campylobacter-like organism, has been designated an emerging pathogen because of its newly recognized ability to cause diarrheal illness in both humans and animals and its presence in the human food supply. Because there is no standard isolation method for its detection, the true occurrence of this pathogen is largely unknown. In addition, the lack of a standardized isolation protocol limits the ability of investigators to compare field data. Arcobacter has been detected in whole muscle and ground pork at various levels by two different isolation methods (those of deBoer and Collins). In this study, these methods were tested along with the Johnson-Murano (JM) method, developed in our laboratory. The sensitivity of each method was tested for ground pork inoculated with Arcobacter butzleri and Arcobacter cryaerophilus IA at levels of 10(4), 10(3), 10(2), and 10(1) CFU/g. Controls included tubes with uninoculated pork and broth tubes without pork. All samples that were morphologically similar to Arcobacter were analyzed by Gram staining and by catalase and oxidasereactions. Presumptive positive samples were confirmed by the polymerase chain reaction. The JM method was determined to be the most sensitive, detecting A. butzleri down to a level of 10(1) CFU/g in 100% of the samples and detecting A. cryaerophilus IA at a level of 10(1) CFU/g in 75% of samples. In a pure buffer system, the Collins method was as effective as the JM method in isolating both organisms to levels of 10(1) cells per g.     

Prevalence and Distribution of Arcobacter spp. in Raw Milk and Retail Raw Beef

A total of 106 beef samples which consisted of local (n = 59) and imported (n = 47) beef and 180 milk samples from cows (n = 86) and goats (n = 94) were collected from Selangor, Malaysia. Overall, 30.2% (32 of 106) of beef samples were found positive for Arcobacter species. Imported beef was significantly more contaminated (46.80%) than local beef (16.9%). Arcobacter butzleri was the species isolated most frequently from imported (81.8%) and local (60%) beef, followed by Arcobacter cryaerophilus in local (33.3%) and imported (18.2%) beef samples. Only one local beef sample (10%) yielded Arcobacter skirrowii. Arcobacter species were detected from cow's milk (5.8%), with A. butzleri as the dominant species (60%), followed by A. cryaerophilus (40%), whereas none of the goat's milk samples were found positive for Arcobacter. This is the first report of the detection of Arcobacter in milk and beef in Malaysia.     

Detection and diversity of various Arcobacter species in Danish poultry

The prevalence and diversity of different Arcobacter spp. in various poultry species in Denmark were investigated using cultural and multiplex PCR methods. A pool of three fresh droppings obtained at the production site from 70 broiler chicken flocks aged 4-5 weeks was examined. In addition, pools of 10 cloacal swabs taken at the abattoir prior to stunning from each of 15, and 37 duck and turkey flocks, respectively, were analyzed. Thirty fresh broiler chicken carcasses and 29 cloacal swabs from the respective viscera were also examined at the abattoir. Finally, 10 caecal and 10 cloacal swabs from ducks at the abattoir were analyzed individually. In total, 85 Arcobacter isolates were obtained. Of these 45, 20 and 7 were identified as Arcobacter butzleri, Arcobacter cryaerophilus and Arcobacter skirrowii, respectively, using a multiplex PCR. Interestingly, some chicken isolates of A. butzleri showed urease activity, and 6 out of seven A. skirrowi isolates were unable to hydrolyse indoxyl acetate. All chicken carcasses examined were found positive for A. butzleri and/or A. cryaerophilus, whereas 21 (72%) of the 29 chicken cloacal swabs were positive for either A. butzleri (13) or A. cryaerophilus (9). Three (4.3%) out of 70 chicken flocks analyzed were positive only for A. cryaerophilus. Of the ten ducks examined individually, 7 carried A. skirrowii and/or A. cryaerophilus in their cloacae. None of the respective caecal samples were positive. Of the remaining 15 duck flocks, 11 (73%) were positive for A. cryaerophilus (7), A. butzleri (2) or A. skirrowii (2). Four (11%) of the 37 turkey flocks analyzed harboured either A. butzleri or A. cryaerophilus. The carriage rate of Arcobacter was higher in live ducks than those of live broiler chickens and turkeys in the present study. In addition, chicken carcasses slaughtered in Denmark were found to be contaminated with Arcobacter. The presence of Arcobacter spp. both on chicken carcasses and in poultry intestine may be of significance to human health.     

A comparison of three methods for the isolation of Arcobacter spp. from retail raw poultry in Northern Ireland

Recent evidence suggests that arcobacters, especially Arcobacter butzleri, are potential foodborne pathogens, but standardized detection methods have yet to be established. A study was undertaken to determine which of three isolation methods was the most effective for the isolation of Arcobacter spp. from fresh raw poultry. Methods 1 was microaerobic and involved a membrane filtration step followed by plating onto blood agar. Method 2 was also microaerobic and involved enrichment and plating media containing a five-antibiotic cocktail. Method 3 was aerobic and was based on enrichment in a charcoal-based broth containing two antibiotics. Retail poultry samples (n = 50) were obtained from supermarkets in Northern Ireland; the European Community license number was recorded to ensure sample diversity. Presumptive arcobacters were identified using genus-specific and species-specific primers. Methods 1 resulted in the lowest recovery of arcobacters (28% of samples positive). The detection rate for method 2 (68%) was higher than that for method 3 (50%), but the difference was not significant (P > 0.05). Modification of method 3 by plating the enrichment broth at 24 h, as well as at 48 h, increased recovery to 68%. Use of methods 2 and 3 together increased the number of positive samples detected by approximately 25% compared with use of either method alone. A. butzleri was the most commonly isolated species using all methods. Method 3 detected Arcobacter cryaerophilus in more samples (n = 3) than did method 1 and 2 (n = 1). Arcobacter skirrowii was detected by only method 3 (n = 1). In terms of sensitivity, ease of use, and diversity of species recovered, modified method 3 was the overall method of choice.     

Determination of the occurrence of Arcobacter butzleri in beef and dairy cattle from Texas by various isolation methods

Arcobacter butzleri is a pathogenic bacterium that has been found in dairy cattle, pigs, poultry, and humans. As of this writing, there are no data on the incidence of A. butzleri in beef cattle. Given the differences in rearing practices used for feedlot cattle and those used for dairy cattle, differences in the incidences of this organism in various types of cattle may also exist. Numerous culture methods have been used to isolate A. butzleri, but there are few data on the comparative efficacies of these methods. The objectives of this study were to determine the incidence of A. butzleri in cattle from Texas and to compare the effectiveness levels of the Johnson-Murano (JM) method (consisting of enrichment in JM broth followed by plating on JM agar) and the Collins method (consisting of enrichment in EMJH-P80 broth followed by plating on Cephalothin, Vancomycin, and Amphotericin B [CVA] agar) in the isolation of this organism. Fifty cattle each from two feedlots, a dairy, and a stocker yard were sampled. Fecal swabs were obtained from cattle, and each sample was cultured by the JM method, the Collins method, and combinations of the two methods with the broth of one method being used with the agar of the other. Polymerase chain reaction was used to identify the isolates for confirmation of A. butzleri. Samples from 18 of 200 cattle tested positive for A. butzleri. This organism was detected by the JM method in 4.5% of the samples and by the Collins method in 2.5% of the samples. An incidence of 4.0% was found when JM broth was used with CVA agar, while no samples tested positive for A. butzleri when EMJH-P80 broth was used with JM agar.     

Prevalence of Arcobacter spp. in raw milk and retail raw meats in Northern Ireland

A 1-year study was undertaken to determine the prevalence of Arcobacter spp. in raw milk and retail raw meats on sale in Northern Ireland. Retail raw poultry samples (n = 94), pork samples (n = 101), and beef samples (n = 108) were obtained from supermarkets in Northern Ireland, and raw milk samples (n = 101) were kindly provided by the Milk Research Laboratory, Department of Agriculture and Rural Development, Belfast, Northern Ireland. Presumptive arcobacters were identified by previously described genus-specific and species-specific PCR assays. Arcobacter spp. were found to be common contaminants of retail raw meats and raw milk in Northern Ireland. Poultry meat (62%) had the highest prevalence, but frequent isolations were made from pork (35%), beef (34%), and raw milk (46%). Arcobacter butzleri was the predominant species isolated from retail raw meats and was the only species isolated from raw milk samples. Arcobacter cryaerophilus was detected less frequently, and Arcobacter skirrowii was detected only as a cocontaminant. To our knowledge, this is the first report of Arcobacter spp. prevalence in a diverse range of products of animal origin in Northern Ireland.