Detection of Pathogenic Yersinia enterocolitica in Meat using Real-Time PCR

Yersinia enterocolitica is an important zoonosis, which can cause disease in humans and animals. The culture methods available for detection of Y. enterocolitica in food samples are time-consuming and seldom successful. Using DNA-based methods, like PCR, this pathogen can be detected more rapidly and with greater sensitivity. The aim of this study was to establish a rapid and accurate real-time PCR method to detect pathogenic Y. enterocolitica in pork. The chromosomal ail−gene, which is only present in pathogenic Y. enterocolitica strains, was used as DNA target for the 5' nuclease PCR assay. The probe was labelled at the 5' end with the fluorescent reporter dye (FAM) and at the 3' end with the quencher dye (TAMRA). A 2-step protocol with 45 cycles was used in a multicolour real-time PCR detection system. A Ct value over 40 indicated a negative result. The DNA extraction procedure for the natural samples was rapid and simply. This qualitative real-time PCR method was shown to be specific and sensitive. Detection rate of ail-positive Y. enterocolitica in 200 pig tonsils was 88 % and 35 % with PCR and culture methods, respectively. When 100 raw pork samples were studied, 7 were positive with PCR and all were culture negative. Received: January 30, 2007; Received in revised form: February 27, 2007; Accepted: February 28, 2007.     

A real-time PCR for Detection of Pathogenic Yersinia enterocolitica in food combined with an Universal Internal Amplification Control System

A real-time PCR system with an internal amplification control was developed for detection of pathogenic Yersinia) enterocolitica in food samples. The chromosomally encoded ail gene was chosen as PCR target. Sequences of plasmid pUC19 served as target for the internal amplification control. The method was validated in combination with sample enrichment in PSB and TSB broth using different food matrices spiked with Y. enterocolitica and naturally contaminated slaughterhouse samples. The results of the real-time PCR with internal control were verified by the cultural method according to EN ISO 10273:2003. The sensitivity of the real-time PCR with internal control is about 5 genome copies per reaction. Artificial contamination of food samples resulted in a detection level of 5 cfu per 25 g Y. enterocolitica in food samples. 100% of porcine tonsils and about 22% meat from pig heads were contaminated. The screening of samples by PCR prior to cultural analysis allows focusing on positive samples in routine analysis. This could result in a higher detection rate by cultural analysis.     

Prevalence of pathogenic Yersinia enterocolitica and Yersinia pseudotuberculosis in wild boars in Switzerland

Between October 2007 and March 2008, 153 wild boars shot in the Canton of Geneva in Switzerland were sampled. Fifty-one percent of the animals were males and 49% were females. The age of most (81%) animals varied between 6 months and 2 years. Prevalence of enteropathogenic Yersinia in tonsils and faeces was studied using culture and PCR methods and in tissue fluid of tonsils using an ELISA system. Prevalence of anti-Yersinia antibodies in tissue fluid was 65%. Detection rate of enteropathogenic Yersinia in tonsils of 153 wild boars by real-time PCR was 44%. Ail-positive Yersinia enterocolitica and inv-positive Yersinia pseudotuberculosis were detected in 35 and 20% of the animals, respectively. Both species were detected in 10% of the animals. Isolation rate of enteropathogenic Yersinia was low; ail-positive Y. enterocolitica and inv-positive Y. pseudotuberculosis were found in 9 and 3% of the animals, respectively. Prevalence was shown to be significantly higher in tonsils than in faeces. Furthermore, females were more commonly positive than males. This study shows that the prevalence of enteropathogenic Yersinia is high and both enteropathogenic Y. enterocolitica and Y. pseudotuberculosis are common findings in tonsils of wild boars in Switzerland.     

Inactivation of Yersinia enterocolitica by pulsed electric fields

The influence of growth conditions, treatment medium characteristics and PEF process parameters on the lethal effect on Yersinia enterocolitica of pulsed electric fields (PEF) treatments in batch has been investigated. Growth phase, temperature of growth, pH, conductivity of the treatment medium, pulse width and frequency of pulses did not influence the sensitivity of Y. enterocolitica to PEF. However, an A_w decrease from >0.99 to 0.93 of the treatment medium increased the PEF resistance of Y. enterocolitica with 3.5 log₁₀ cycles after a treatment of 22 kV/cm, 800 μs and 880 kJ/kg. Inactivation of Y. enterocolitica increased with the field strength, treatment time and total specific energy up to a maximum of 6 log₁₀ cycles after 28 kV/cm, 2000 μs and 3559 kJ/kg. A nonlinear relationship was found among the survival fraction and the treatment time or the specific energy that was accurately described by a mathematical model based on the Weibull distribution. The inactivation of Y. enterocolitica by PEF was characterized by maximum field strength thresholds. Above these thresholds, specific energy necessary to obtain a given level of inactivation scarcely decreased by increasing the electric field strength, and inactivation of Y. enterocolitica only depended on the specific energy applied.     

温州市食品中小肠结肠炎耶尔森氏菌分布及分子分型研究

目的 了解温州市食品中小肠结肠炎耶尔森氏菌的分子分型及分布特征。方法 4 ℃增菌后用选择性培养基对食品中的小肠结肠炎耶尔森氏菌进行分离鉴定,分析阳性菌株的生物型、血清型、毒力基因型、耐药性和脉冲场凝胶电泳（PFGE）分子型别。结果 采集6类食品,共676份样品,其中68份样品检出69株小肠结肠炎耶尔森氏菌,检出率为10.1%（68/676）。调理肉制品检出率最高（20.5%,9/44）,其次为速冻食品（17.2%,11/64）。95.7%（66/69）的分离株为生物1A型,生物血清型以1A/O∶5（29.0%）为主,其次为1A/O∶8（14.5%）;88.4%（61/69）的菌株仅携带ystB基因,检出1株4/O∶3型菌株携带毒力基因ail、ystA、yadA、virF。分离株对14种抗菌药物的敏感率达到94%以上。32株血清已分型菌株,分为29种PFGE带型。结论 温州市食品中存在一定程度的小肠结肠炎耶尔森氏菌污染,且检出致病性小肠结肠炎耶尔森菌,菌株耐药率处于较低水平,分子分型提示菌株呈高度遗传多态性。     

Pathogenic Yersinia enterocolitica 2/O:9 and Yersinia pseudotuberculosis 1/O:1 strains isolated from human and non-human sources in the Plateau State of Nigeria

Foodborne yersiniosis, caused by enteropathogenic Yersinia, especially Yersinia enterocolitica, is an important cause of diarrhea in developed countries, especially in temperate zones. Since studies concerning the presence of enteropathogenic Yersinia in humans and foods are rare in developing countries and tropical areas, human and non-human samples were studied in Plateau state of Nigeria to obtain information on the epidemiology of Y. enterocolitica and Yersinia pseudotuberculosis. Surprisingly, ail-positive Y. enterocolitica and inv-positive Y. pseudotuberculosis were isolated in Plateau state of Nigeria from several samples of human and non-human origin. Bioserotype 1/O:1 was the only Y. pseudotuberculosis type found. Y. enterocolitica belonging to bioserotype 2/O:9 was the dominating type found in most samples. Bioserotype 4/O:3 was isolated only from one pig and one sheep. Using PFGE, 5 genotypes were obtained among 45 Y. enterocolitica 2/O:9 strains with NotI, ApaI and XhoI enzymes and 3 among 20 Y. pseudotuberculosis 1/O:1 strains with NotI and SpeI enzymes. All human Y. pseudotuberculosis 1/O:1 strains were indistinguishable from pig, sheep or food strains. The dominating genotype of Y. enterocolitica 2/O:9 strains among humans was also found among strains isolated from pig, fermented cow milk and traditional intestine pepper soap samples.     

A model for lactic acid-induced inhibition of Yersinia enterocolitica in mono- and coculture with Lactobacillus sakei

Most traditional predictive models for growth of foodborne pathogenic (or spoilage) organisms focus on single species behaviour. This approach may lead to a significant discrepancy between model predictions and reality, since the (potential) influence of the background flora is neglected. In this paper, a specific type of multiple species interaction is considered, namely, a pathogenic organism in the presence of a microbial antagonist, where the latter species inhibits the pathogen's growth through lactic acid production. As an experimental case study, growth curves of Yersinia enterocolitica in mono- and coculture with Lactobacillus sakei were generated in duplicate in a modified brain–heart infusion medium. A complete factorial design was applied to assess the impact of temperature (4 levels) and inoculum ratio pathogen/antagonist (6 levels) on the Y. enterocolitica growth. Based on a set of formulated model requirements, a novel model was developed, in which the experimentally observed inhibition effect, i.e., an (early) induction of the stationary phase in the pathogen's growth curve, is explicitly related to the lactic acid production curve. Experimental data and model predictions show good agreement.     

肉及肉制品中6种致病菌的GeXP多重PCR检测

利用多重基因表达(GenomeLabTM eXpress Profiling,GeXP)遗传分析系统建立一种可单管一次同时检测大肠杆菌O157:H7、金黄色葡萄球菌、志贺氏菌、副溶血性弧菌、单核细胞增生李斯特菌和沙门氏菌6种肉及肉制品中致病菌的多重聚合酶链式反应(polymerase chain reaction,PC...     

Real-Time TaqMan PCR for Rapid Detection and Quantification of Coliforms in Chilled Meat

Coliforms are a major group of bacteria associated with spoilage of refrigerated pork. It is necessary to develop an efficient and fast method to detect total coliforms in chilled pork because the traditional methods are laborious and time-consuming. In this study, a quantitative real-time polymerase chain reaction (qRT-PCR) targeting the lacZ gene was developed for rapid enumeration of coliforms from chilled pork. Of the 106 strains tested, 35 coliform strains and one Shigella sonnei strain were correctly identified compared with 70 control strains which failed to amplify. Detection sensitivity was 112 CFU/g. The assay was able to detect and accurately quantify the total coliforms in chilled meat within 2 h without preenrichment. The results indicate that the real-time PCR is an efficient and time-saving method that could be adopted by the meat industry to detect coliforms to replace the traditional most probable number and the rapid coliform count plate methods.     

食品接触材料中3种致病菌的多重荧光PCR检测

建立并优化食品接触材料中金黄色葡萄球菌、沙门氏菌和副溶血性弧菌3种致病菌的多重实时荧光聚合酶链式反应(polymerase chain reaction,PCR)检测方法,评价此方法的特异性和灵敏度,并模拟阳性样品进行检测。结果显示,该方法特异性良好,检测灵敏度均能达到10~3 CFU/mL,为食品接触材料的微生物检测提供技术保障。     

实时荧光定量PCR法快速检测食品中大肠埃希菌

目的应用实时荧光定量PCR技术,结合3~5 h的前增菌处理,建立食品中大肠埃希菌的快速、灵敏、定量的检测方法。方法以大肠埃希菌(ATCC 25922)为参考菌株,对培养基和培养温度进行优化,选择最佳的前增菌培养条件。将不同浓度的参考菌株和样品分别接种前增菌液中培养3~5 h。采用Triton-X 100提取增菌后的DNA,实时荧光定量PCR扩增大肠埃希菌特异性片段。所得Ct与对应的原始(增菌前)参考菌株的浓度,建立标准曲线,计算样品中大肠埃希菌的数量。结果纯培养模式下,经过3、4和5 h的前增菌后,标准曲线具有很好的线性,r2分别为0.996、0.992和0.991,对应的检测限为136、14和1.4 cfu/100 ml;含杂菌培养模式下,NB和EC肉汤42.0℃增菌4 h后,建立的标准曲线r2分别为0.972和0.978。在不同食品中该方法的加标回收率为74.0%~174.0%。结论 3~5 h的前增菌实时荧光定量PCR方法可以快速、灵敏、定量地检测食品中活的大肠埃希菌。     

肉制品中致病菌的在线检验技术

细菌性食物中毒是食物中毒的重要因素。以前国内检测食品中致病菌主要采用传统的培养方法,操作繁琐,检测时间较长。现在一些致病微生物的快速检测技术得到了迅速地发展。本文主要对肉制品中致病菌的检验进行综述,如免疫学中的酶免疫分析、荧光免疫分析等;分子生物学中的核酸探针和聚合酶链反应的检测技术等,旨在对肉制品中致病菌的在线检验技术做出较全面的总结,为食品安全提供理论依据。     

实时PCR技术在羊亚科肉类鉴定中的应用

根据线粒体cyt b基因的种间差异,建立羊亚科、绵羊、山羊肉类的实时荧光聚合酶链式反应检测技术,对方法进行系统的优化与完善,并进行特异性、灵敏度与扩增效率、检出限、试剂一致性分析与适用性考察,同时邀请5家国内权威机构对方法进行复核。结果表明,方法的重复性好、适用性广,可用于不同基质的肉制品检测,扩增效率达95%以上,用于羊肉制品的动物源性成分检测准确率达100%,适于推广使用。     

肉品中食源性致病菌检测技术研究进展

肉品易受食源性致病菌污染,是食品安全监管的重点.借由现代检测技术快速可靠的鉴别及检验肉品中的食源性致病菌具有重要的现实意义.本文在总结肉品典型致病菌污染现状的基础上,重点综述了分子诊断法、免疫分析法、光谱检测法及电子鼻检测法等致病菌检测技术研究进展,在分析现有技术存在主要问题的同时,对其未来发展方向进行展望,旨在为肉品...     

肉及肉制品中沙门氏菌活细胞的PCR检测研究

将荧光染料叠氮溴化丙锭(propidium monoazide,PMA)与普通聚合酶链式反应(polymerase chain reaction,PCR)结合,通过对PMA的曝光时间、浓度进行优化,确定PMA-PCR区别死活细胞的最佳条件,并制作活细胞定量标准曲线,建立肉及肉制品中沙门氏菌活细胞的PMA-PCR检测方法。结果表明：使插入死细胞DNA中的PMA活化并且光解溶液中游离PMA的最佳曝光时间为15min;不抑制沙门氏菌活细胞DNA扩增的最大PMA质量浓度为10μg/mL;完全抑制热致死细胞DNA扩增的最小PMA质量浓度为4μg/mL。经PMA处理,含有不同比例的沙门氏菌热致死细胞和活细胞的混合液中活的沙门氏菌能够通过PCR被选择性的检测,最小检测限为20CFU/PCR。而且,经研究发现在20～2×105CFU/PCR范围内,电泳条带相对荧光强度与活细胞数的对数具有线性关系。采集30份肉及肉制品样品,利用PMA-PCR方法检测出两份生肉样品中存在沙门氏菌,经过6h的富集培养后的活菌浓度分别为2.5×103CFU/mL和3.4×103CFU/mL。     

4种动物源性成分多重real-time PCR检测方法的建立及其在驴肉制品检测中的应用

建立一种同时快速检测驴、马、猪及鸭源性成分的四重实时聚合酶链式反应(real-time polymerase chain reaction,real-time PCR)方法。分别以4种源性成分的Nad5、ATpase6、ATP8、cytb基因为靶基因设计特异性引物和Taq Man探针,以18S r RNA基因为内参基因,建立多重real-time PCR方法,并对该方法进行方法学验证,同时对不同掺入比例模拟样品、不同加工工艺模拟样品和实际驴肉样品进行检测。结果显示,该方法具有高通量、特异性强、灵敏度高等优点。当Ct值≤35.0时,方法对16种非目标源性具有良好特异性;灵敏度可检测到质量浓度为2×10^-4 ng/μL的模板DNA;对生肉的检出限为肉含量的0.001%,对熟肉制品的检出限为肉含量的0.01%;对100份实际样品进行检测,结果与标准方法一致,说明建立的多重real-time PCR法可用于肉及肉制品中常见掺假源性成分的检测。     

Validation of a 1-Day Analytical Diagnostic Real-Time PCR for the Detection of Salmonella in Different Food Meat Categories

Foodborne disease caused by Salmonella represents a worldwide public health problem. In Europe, salmonellosis is still the second most commonly recorded zoonosis. Since the standard culture method for detecting Salmonella (ISO 6579:2002) requires up to 5 days to produce results, the need to develop rapid methods represents an important issue for the authorities and the producers. The aim of the present study was the in-house validation, according to ISO 16140, of an open-formula diagnostic real-time PCR for the detection of Salmonella in all the different meat categories reported in the EU Regulations relative to microbiological criteria for food safety. The assay employed specific primers and a probe target within the ttrRSBCA locus, which allows the tetrathionate respiration in Salmonella. Selectivity, relative accuracy, relative sensitivity and relative specificity were established by testing 110 bacterial strains and 175 various edible meat samples. Results showed 100 % selectivity, 100 % relative accuracy, 100 % relative sensitivity and 100 % relative specificity of the real-time PCR when compared to the standard culture method used as reference. In addition, in order to minimize the effect of the competitive micro-flora naturally present on meat samples, a highly nutritious and selective commercial medium (ONE Broth Salmonella, Oxoid) was evaluated in comparison with the classical non-selective pre-enrichment broth (buffered peptone water). Results demonstrated that the ONE Broth Salmonella medium increases the growth of Salmonella in the presence of competitive micro-flora.     

食品沙门氏菌实时荧光PCR快速检测方法建立

根据Genbank提供的沙门氏菌ttrBCA基因序列设计引物和探针,建立了食品沙门氏菌实时荧光PCR快速检测方法。结果显示,该方法只对沙门氏菌基因呈阳性反应,而对其它常见非阳性菌株(志贺氏菌、金黄色葡萄球菌、大肠杆菌、奇异变形杆菌、普通变形杆菌、绿脓杆菌、单增李斯特氏菌、阴沟肠杆菌、副溶血性弧菌)基因组DNA均呈阴性反应,对模拟添加沙门氏菌样品检测,检测低限为240cfu/mL沙门氏菌的DNA,检测食品样品增菌液,仅需约3h,结果表明,该方法适用于食品样品的快速检测。     

应用RT-PCR技术定量检测发酵乳中Lactobacillus plantarum活菌数

根据发酵乳中常见乳酸菌种的16S rRNA基因序列设计了L.plantarum的种属特异性引物,应用该引物对植物乳杆菌的模式菌株L.plantarum ATCC14917、L.plantarum非同源性对照菌株及采自西藏地区的自然发酵乳样品进行了种属特异性PCR检测,并以样品RNA转录的cDNA为模板,对检出含有L.plantarum的发酵乳样品进行了Real-Time PCR定量检测。通过Real-Time PCR图谱分析,准确地检测出了该样品中L.plantarum活菌数的含量为6.6lgcfu/mL。结果表明该方法能够简便、快速地检测出发酵乳中是否含有L.plantarum,并能够对其活菌数进行准确地定量,对发酵乳的生产和监管提供了重要的理论和实践依据。