Isolation of Yersinia enterocolitica from raw cow milk in Argentina

A survey was carried out to determine the presence of Yersinia enterocolitica in raw cow milk from producers in Buenos Aires, Argentina. From March to June 1984, 18 pooled- and 93 individual-producer samples were examined by two enrichment procedures. Y. enterocolitica was isolated from 6 samples (5.5%). Eleven samples (9.9%) yielded Yersinia frederiksenii and 4 (3.6%) Yersinia intermedia. All the isolates of Y. enterocolitica belonged to biotype 1 of Wauters. Two serotypes, 0:5 and 0:10,34 were identified. The phage types were divided into Xo and Xz. The strains were avirulent by calcium dependence, autoagglutination and mouse i.p. injection tests.     

Development of a multiplex PCR for rapid detection of verocytotoxin-producing Escherichia coli O26 in raw milk and ground beef

Verocytotoxin-producing Escherichia coli (VTEC) O26 is an emergent pathotype that has caused an increasing number of sporadic cases and outbreaks of gastroenteritis, hemorrhagic colitis, and hemolytic uremic syndrome in the United States and Europe. Many cases are associated with the consumption of milk and undercooked or fermented meats. The stx(2) strains of VTEC O26 seem to be more likely to cause human infections than isolates expressing only stx(1). The isolation and identification of VTEC O26 from foods is labor intensive and time-consuming. We developed a multiplex PCR (M-PCR) assay for the identification and characterization of E. coli O26 VTEC and its detection in raw milk and ground beef. The method is based on the amplification of the wzx, stx(1), and stx(2) genes for the simultaneous detection of the O26 antigen and verocytotoxin types 1 and 2. This M-PCR assay had a sensitivity of 10(8) CFU/ml when applied to a bacterial suspension and of 10(6) CFU/ml or g when applied to both inoculated milk and minced beef samples. This M-PCR assay also was highly specific, and results were consistently negative for negative controls (nonpathogenic E. coli strains, uninoculated milk and beef samples, and samples inoculated with the nontarget microorganisms). This method could be used for the rapid detection of E. coli O26 VTEC from foods and for the rapid identification and characterization of clinical and environmental isolates.     

Development of a multiplex real-time PCR assay for the detection of ruminant DNA

The U.S. Food and Drug Administration (FDA) has previously validated a real-time PCR-based assay that is currently being used by the FDA and several state laboratories as the official screening method. Due to several shortcomings to the assay, a multiplex real-time PCR assay (MRTA) to detect three ruminant species (bovine, caprine, and ovine) was developed using a lyophilized bead design. The assay contained two primer or probe sets: a "ruminant" set to detect bovine-, caprine-, and ovine-derived materials and a second set to serve as an internal PCR control, formatted using a lyophilized bead design. Performance of the assay was evaluated against stringent acceptance criteria developed by the FDA's Center for Veterinary Medicine's Office of Research. The MRTA for the detection of ruminant DNA passed the stringent acceptance criteria for specificity, sensitivity, and selectivity. The assay met sensitivity and reproducibility requirements by detecting 30 of 30 complete feed samples fortified with meals at 0.1 % (wt/wt) rendered material from each of the three ruminant species. The MRTA demonstrated 100 % selectivity (0.0 % false positives) for negative controls throughout the assessment period. The assay showed ruggedness in both sample selection and reagent preparation. Second and third analyst trials confirmed the quality of the written standard operating procedure with consistency of results. An external laboratory participating in a peer-verification trial demonstrated 100 % specificity in identifying bovine meat and bone meal, while exhibiting a 0.03 % rate of false positives. The assay demonstrated equal levels of sensitivity and reproducibility compared with the FDA's current validated real-time PCR assay. The assay detected three prohibited species in less than 1.5 h of total assay time, a significant improvement over the current real-time assay. These results demonstrated this assay's suitability for routine regulatory use both as a primary screening tool and as a confirmatory test.     

食品中3种致病菌多重PCR检测方法的建立及初步应用

建立用多重聚合酶链式反应(Multiplex polymerase chain reaction,mPCR)同时检测食品中沙门氏菌、金黄色葡萄球菌和单核细胞增生性李斯特菌的方法。以沙门氏菌的gyrB基因、单核细胞增生性李斯特菌的gyrB基因、金黄色葡萄球菌的coa基因作为目的基因,分别设计3对引物,通过优化反应体系,建立3种致病菌的多重PCR检测体系。采用单重PCR检测时,灵敏度可达0.423ng/mL(沙门氏菌)、2.5ng/mL(金黄色葡萄球菌)和0.36ng/mL(单核细胞增生性李斯特菌);而采用三重PCR检测时,灵敏度较单重PCR有所下降,分别为2.43ng/mL(沙门氏菌)、2.5ng/mL(金黄色葡萄球菌)、3.6ng/mL(单核细胞增生性李斯特菌)。初步建立能同时、快速、灵敏地检测食品中沙门氏菌、金黄色葡萄球菌和单核细胞增生性李斯特菌的三重PCR方法。     

Development of a real-time PCR assay for the detection of cow and donkey milk

A real-time PCR allelic discrimination TaqMan assay based on the analysis of a single nucleotide polymorphism enabling the differentiation of cow (Bos taurus) and donkey (Equus asinus) milk was developed. Specific primers and probes were designed on the mitochondrial cytochrome c oxidase subunit I gene. The primers were designed upstream and downstream the chosen diagnosis site in a conserved region. Two probes were designed to specifically hybridise to B. taurus and E. asinus sequences. The test allowed the discrimination of bovine and donkey DNA in all blood and pure milk samples giving an unambiguous result plot of rapid and easy interpretation. The detection threshold was 2?% of cow milk in donkey milk. The applicability of the method to matrices containing degraded DNA was demonstrated by analysing samples of raw donkey and cow milk autoclave-treated (121?°C for 15?min). Finally, the assay when applied to milk samples collected from the retail trade has confirmed the species indicated in the label. Furthermore, the assay represents a potentially valuable diagnostic tool for species identification in dairy products for allergic people.     

Enumeration and confirmation of Aeromonas hydrophila, Aeromonas caviae, and Aeromonas sobria isolated from raw milk and other milk products in Northern Greece.

A total of 138 raw cow's and 57 raw ewe's milk samples; 80 pasteurized cow's milk samples; 39 Anthotyros cheese, 36 Manouri cheese, and 23 Feta cheese samples; and 15 rice pudding samples were examined for the presence and any countable population of Aeromonas species. Twenty-two (15.9%) of the 138 cow's milk samples analyzed were contaminated with A. hydrophila. In 13 of these samples, populations of 3.0x10(2) to 5.0x10(3) CFU/ml were counted in starch ampicillin agar (SAA). Eighteen cow's milk samples (13.0%) were contaminated with A. caviae, and in eight of these samples, populations of 2.0x10(2) to 3.0x10(3) CFU/ml were counted in SAA. Five cow's milk samples (3.6%) were contaminated with A. sobria, and in two of these samples, populations of 2.5x10(3) and 5.0x10(3) CFU/ml were counted in SAA. Eleven cow's milk samples (7.9%) were contaminated with other Aeromonas spp. not classified. Eight (14.0%) of the 57 ewe's milk samples analyzed were contaminated with A. hydrophila. In these samples, populations of 5.0x10(2) to 5.0x10(3) CFU/ml were counted in SAA. Six ewe's milk samples (10.5%) were contaminated with A. caviae, and populations of 1.5x10(2) to 1.0x10(3) CFU/ ml were counted in SAA. Two ewe's milk samples (3.5%) were contaminated with A. sobria, and populations counted in SAA were 5.0x10(2) and 1.0x10(3) CFU/ml. Four samples (7.0%) were contaminated with other Aeromonas spp. not classified. A. hydrophila was recovered in 4 (10.2%) and 3 (8.3%) of the Anthotyros and Manouri cheese samples analyzed, respectively, but no countable populations were noted in SAA. None of the pasteurized milk, Feta cheese, and rice pudding samples yielded Aeromonas spp. The results of this work indicate that motile Aeromonas are common in raw milk in Greece. Also, the presence of A. hydrophila in the whey cheeses Anthotyros and Manouri indicates that postprocessing contaminations of these products with motile Aeromonas may occur during production.     

Yersinia enterocolitica in milk and dairy products

Yersinia enterocolitica was first recognized during the 1960's as an important human enteropathogen. The species as later redefined includes both pathogenic and nonpathogenic forms. Pathogenic strains that retain the virulence plasmid can be identified in several animal models and four indirect tests (calcium dependency, autoagglutination, Congo red uptake, serological detection of outer membrane antigen) and by tissue culture assay, serotype, and biotype. Y. enterocolitica and related bacteria have frequently been isolated from raw milk, but none of the isolates, with the possible exception of serotype 05,27, are recognizable as pathogens. Under normal circumstances Y. enterocolitica does not survive pasteurization. If introduced into pasteurized milk, it can grow well at refrigeration temperatures. Two outbreaks of yersiniosis have occurred that involved pasteurized milk. Pigs, which frequently carry pathogenic Y. enterocolitica in their throat, were the probable source in one of these outbreaks. The most rapid enrichment procedure available for isolation of Y. enterocolitica requires 6 d. No isolation method is available for selective isolation of pathogenic Y. enterocolitica in the presence of related bacteria common in milk and other foods.     

Evaluation of a 5'-nuclease (TaqMan) assay for the detection of virulent strains of Yersinia enterocolitica in raw meat and tofu samples

Culture methods for detecting virulent Yersinia enterocolitica require selective enrichment and a series of confirmatory tests that are time-consuming, costly, and laborious. The objective of this study was to evaluate a fluorogenic 5'-nuclease assay for detecting the enterotoxin yst gene of virulent Y. enterocolitica in pure cultures, inoculated ground pork samples, and naturally contaminated food samples. These results were then compared with "gold standard" methods recommended by the U.S. Food and Drug Administration in the Bacteriological Analytical Manual for detecting pathogenic Y. enterocolitica. The 5'-nuclease assay was able to identify the organism in 100% of the repetitions when 10(2) CFU/ml or more organisms were present in pure cultures and 10(3) CFU/g or more organisms were present in ground pork. Similar recovery efficiency on cefsulodin-irgasan-novobiocin (CIN) agar plates was only evident when 10(5) CFU/ml or more organisms were present in pure culture and 10(6) CFU/g or more organisms were present in inoculated ground pork. The 5'-nuclease assay indicated a contamination rate of 35.5% (94/265) in various meats and tofu, whereas the CIN plating method indicated a contamination rate of 28.3% (75/265). This resulted in 100% sensitivity and 64.5% specificity for the 5'-nuclease assay when compared with the standard culture recovery method. Only 75% (60/80) of the Yersinia spp. isolated on CIN was identified as containing a virulence plasmid by autoagglutination and crystal violet binding tests. These results indicate that the true rate of contamination of virulent Y. enterocolitica in pork and other processed meats and foods is being underestimated using current detection methods. This study demonstrates the potential of the 5'-nuclease assay for rapidly and specifically detecting virulent Y. enterocolitica in processed foods with the added advantage of being an automated detection system with high-throughput capability.     

Prevalence of virulent Yersinia enterocolitica in bulk raw milk and retail cheese in northern-west of Iran

To determine the prevalence of virulent Yersinia enterocolitica, 554 samples consisting of 354 bulk raw milks and 200 traditional cheeses were collected from different parts of Eastern-Azerbaijan province, during a 23-month period from 2008 to 2010. The occurrence of virulent strains of Y. enterocolitica in samples enriched in peptone sorbitol bile broth (PSBB) was evaluated via the detection of attachment invasion locus (ail) gene by PCR. The viability of virulent Y. enterocolitica in the PCR-positive samples was tested using conventional culture method and the isolates were confirmed by the second-phase ail-PCR. According to the results, 8.66% of total samples including 7.62% of bulk raw milks and 10.5% of raw milk cheeses were found ail-positive by PCR method; subsequently Y. enterocolitica was isolated by the culture method and confirmed by the second phase ail-PCR in 2.88% of total samples including 2.26% of raw milks and 4% of cheese samples. It was concluded that, a sample enrichment followed by ail-PCR was more sensitive and robust to detect and distinguish the virulent strains of Y. enterocolitica compared to the conventional culture method.     

Antibacterial activity of smoke wood condensates against Aeromonas hydrophila,Yersinia enterocolitica andListeria monocytogenes at low temperature

The antibacterial activity of four commercial preparations of smoke condensates was tested against three psychrotrophic foodborne pathogens (Aeromonas hydrophila, Yersinia enterocolitica and Listeria monocytogenes). The effects of the recommended concentrations for the use of one dried (S) and three liquid (L1, L2, L3) extracts were examined in tryptic soy broth for 21 days at refrigeration temperature. The results revealed that smoke extracts differed considerably in their ability to inhibit the growth of the investigated pathogens. A. hydrophila was the most susceptible organism. Extracts L1, L2 and S caused a gradual decline in A. hydrophila viable cell counts to reach undetectable levels. Extracts S was bacteriostatic to L. monocytogenes and Y. enterocolitica. Extract L1 reduced populations of these organisms during the experiment. Extract L2 reduced the population of L. monocytogenes. This extract did not affect, however, Y. enterocolitica growth. The least effective extract was L3 which only inhibited the growth of A. hydrophila during the experimental period and did not show any effect against L. monocytogenes and Y. enterocolitica. The compounds and their relative concentrations identified and quantified by gas chromatography/mass spectrometry varied greatly among the analysed condensates. No relation has been shown between the antimicrobial activity and the concentration of phenols of smoke preparations.     

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.     

Comparison of four methods for isolation of Yersinia enterocolitica from raw and pasteurized milk from northern Iran

Four methods for isolation of Yersinia enterocolitica from raw and pasteurized milk from northern Iran were compared. Three hundred and ten raw milk samples were collected from tanks on their arrival at various central dairies, and 40 pasteurized milk samples were collected from tanks on their arrival at a manufacturing plant. Each sample was examined for the presence of Y. enterocolitica by (1) direct culture; (2) enrichment in double-strength buffered peptone water at 4 degrees C for 1 month; (3) enrichment in modified Rappaport medium at room temperature for 72 h after a preenrichment in double-strength peptone water at 4 degrees C for 1 month; and (4) enrichment in a medium containing sucrose, tris (hydroxymethyl) aminomethane, sodium azide, and ampicillin at 28 degrees C for 48 h after a preenrichment in double-strength peptone water at 4 degrees C for 1 month. All samples and enrichments were spread on MacConkey agar plus calcium chloride and Tween 80, Yersinia selective agar, and Hektoen medium plus ampicillin. Five samples (1.6%) of raw milk but no pasteurized milk samples were positive for Y. enterocolitica. No Y. enterocolitica were recovered by methods 1 or 2. Y. enterocolitica were recovered from 2 samples by method 3 followed by culture on Yersinia selective agar, and from 5 samples by method 4 followed by culture on Hektoen medium plus ampicillin. The isolates were biotype 1A or 1B, serotype O:7-13 or O:9 and phage type Xo or Xz. All isolates were resistant to ampicillin and amoxicillin, and sensitive to tetracycline, streptomycin, chloramphenicol, and trimethoprim-sulfamethoxazole.     

环介导等温扩增技术检测小肠结肠炎耶尔森氏菌的研究

采用环介导等温扩增技术(Loop-mediated isothermal amplification,LAMP)快速检测小肠结肠炎耶尔森氏菌(Yersinia enterocolitica)。以小肠结肠炎耶尔森氏菌(AY004311.1)Ail基因序列作为靶序列,设计内、外引物,通过肉眼观察沉淀判断检测结果。结果表明:LAMP检测小肠结肠炎耶尔森氏菌的灵敏度为6.3cfu/mL,人工污染鸡肉的检出限为340cfu/g。PCR检测小肠结肠炎耶尔森氏菌的灵敏度为630cfu/mL,人工污染鸡肉的检出限为3.4×104cfu/g。采用试剂盒法提取DNA,从样品处理到报告结果,LAMP方法耗时2h,PCR方法耗时3h。因此,LAMP检测小肠结肠炎耶尔森氏菌的灵敏度高,耗时短,特异性好,操作简便,无需特殊的仪器设备,适合在我国广大基层实验室开展应用,为快速检测食源性致病菌构建了一个新的技术平台。     

Application of multiplex PCR for monitoring colonization of pig tonsils by Yersinia enterocolitica, including biotype 1A, and Yersinia pseudotuberculosis

A multiplex PCR assay was developed for the detection and differentiation of the Yersinia enterocolitica and Yersinia pseudotuberculosis isolates in both pure bacterial cultures and pig tonsils. The assay was based on the amplification of the ail, inv, yadA, and ystB genes. The PCR products, corresponding to the ail gene and the plasmid-borne yadA gene or only one product corresponding to the ail gene, were detected in Y. enterocolitica 4 biotype isolates. All of the Y. pseudotuberculosis isolates (n=6) tested gave a positive PCR reaction for the inv gene. For all tested Y. enterocolitica 1A biotype isolates (n=31), one product corresponding to the ystB gene was observed. The multiplex PCR assay was used to detect Y. enterocolitica and Y. pseudotuberculosis strains in pig tonsil samples obtained from 80 slaughtered pigs from three different herds. The presence of at least one of the specific PCR amplification products of ail-, ystB-, yadA-, and inv-specific sequences was observed in 11 samples (13.75%). These results of the multiplex PCR assay were compared with the results of conventional, microbiological testing. Y. enterocolitica isolates were cultured from only 3 (3.75%) of the 80 pig tonsils examined. The multiplex PCR assay was shown to be an efficient tool for differentiation between the pYV plasmid-bearing Y. enterocolitica isolates, the plasmidless Y. enterocolitica isolates, the Y. enterocolitica biotype 1A isolates, and the Y. pseudotuberculosis isolates with and without the pYV plasmid in naturally contaminated pig tonsils. This indicates that this assay is useful to control food processing and track the source of contamination.     

A simple and rapid realtime PCR assay for the detection of Shigella and Escherichia coli species in raw milk

Escherichia coli and Shigella spp. are two major bacterial contaminants in raw milk. Effective screening of the two microbes before starting the production process is a critical step for the quality and safety guarantee of the resulting dairy products. This study reported a rapid and simple realtime PCR assay using a ubiquitous primer and probe set targeting the tuf gene for the detection of E. coli and Shigella spp. An internal amplification control (IAC) was also comprised to indicate false-negative results. The duplex realtime PCR assay showed a high efficiency above 96 % and a detection limit <10 cfu per PCR. When artificially contaminated raw milk samples were further evaluated, the assay performed equally as well as the traditionally cultural-based method, and facilitated quantitative detection of the two microbes in the range from ~10² to ~10⁶ cfu mL^?1 raw milk. The detection limit was ~10² cfu mL^?1 raw milk for either or a mixture of the two strains without pre-enrichment step, and could be <10 cfu per 10 mL of raw milk if a pre-enrichment step was added. Considering the detection effectiveness, time-consumption saving, and economical efficiency, the present duplex realtime PCR assay has a great potential in the application in raw milk for assessing their microbiological quality and safety in relation to E. coli and Shigella spp.     

Design and validation of a novel multiplex real-time PCR assay for Vibrio pathogen detection

Three species--Vibrio cholerae, Vibrio parahaemolyticus, and Vibrio vulnificus--account for the majority of vibrio infections in humans. Rapid and accurate identification of Vibrio species has been problematic because phenotypic characteristics are variable within species. Additionally, biochemical identification and confirmation require 2 or more days to complete. Rapid and sensitive molecular techniques for the detection of vibrio pathogens would be useful for the surveillance and management of outbreaks. To facilitate the identification of human-pathogenic species, we designed and validated a highly sensitive, specific, and robust multiplex real-time PCR assay to identify V. cholerae, V. parahaemolyticus, and V. vulnificus using a four-dye configuration in a convenient lyophilized format. Multiple Vibrio strains were sequenced to verify candidate target TaqMan sites. Several individual assays within the multiplex contain multiple primers or probes to ensure detection of polymorphic variants. V. cholerae, V. parahaemolyticus, and V. vulnificus were detected either individually or in mixtures at ≤30 genomic copies. V. cholerae was specifically detected in the presence or absence of Vibrio mimicus. The Vibrio multiplex assay showed 100% specificity to all targets analyzed and no detection of nearest neighbor strains. Each assay exhibited 100% ± 10% efficiency. Multiplex real-time PCR can simplify pathogen detection and reduce costs per test since three species can be analyzed in a single reaction tube. Rapid and accurate detection of pathogenic vibrios in shellfish or seawater samples will improve the microbiological safety of seafood for consumers.     

Development and application of a new multiplex real‐time PCR assay for simultaneous identification of Brucella melitensis,Cronobacter sakazakii and Listeria monocytogenes in raw milk and cheese

Brucella melitensis, Cronobacter sakazakii and Listeria monocytogenes are important foodborne pathogens in milk and milk products, which are responsible for a variety of diseases that pose serious hazards to public health and food safety. The objective of this study was to develop a novel multiplex RTi‐PCR for the detection of B. melitensis, C. sakazakii and L. monocytogenes and to characterise the potential risk of these pathogens in raw milk and cheese. The raw milk (n = 25) and cheese samples (n = 20) were analysed by multiplex RTi‐PCR assay and detected for quantification of the three pathogens. In this study, B. melitensis, C. sakazakii and L. monocytogenes were simultaneously identified using BMEII0466, mms operon and hly as target genes, respectively. The multiplex RTi‐PCR assay that was developed showed good sensitivity and selectivity for the pathogenic microorganisms (r² = 0.986–0.997). Multiplex RTi‐PCR results showed that most of the samples were contaminated with the pathogens screened.     

Evaluation of selective enrichment PCR procedures for Yersinia enterocolitica

Four enrichment PCR protocols for detecting unlysed cells of pathogenic Yersinia enterocolitica were studied. First, the probability of detecting Y. enterocolitica cells of known concentrations by a multiplex PCR assay was determined, and it was found to follow a logistic regression model. From this model, the probability of detecting Y enterocolitica at a specific concentration could be estimated; for example, the detection probability of 10(4) CFU/ml was estimated to be 85.4%. The protocols were evaluated on enrichment cultures inoculated with 10(2) CFU/ml Y. enterocolitica and 10(2)-10(6) CFU/ml of a defined background flora. For each protocol, the time for sample withdrawal and the presence of background flora were studied with respect to PCR detection. The optimal point in time of sample withdrawal was found to be different for each protocol employed. Early detection was favoured by concentrating the target cells, and the most rapid PCR detection of Y. enterocolitica was achieved with enrichment in Yersinia-PCR-compatible-enrichment (YPCE) medium for 3 h at 25 degrees C, followed by a centrifugation prior to PCR analysis. For detection of Y. enterocolitica in the presence of high concentrations (10(6) CFU/ml) of background flora, a long incubation time followed by density centrifugation and a dilution step was most successful. The protocol that gave the most reliable PCR detection in the presence of 10(6) CFU/ml background flora included 24 h incubation in Yersinia-selective-enrichment (YSE) broth at 25 degrees C, followed by Percoll density centrifugation, and a 100 times dilution prior to PCR analysis.     

Taqman荧光实时PCR快速检测原料乳中EPEC

建立可对原料乳中肠致病性大肠埃希氏菌(enteropathogenic Escherichia coli,EPEC)进行快速检测的Taqman光实时PCR方法.以eae致病基因为靶基因,用特异性引物和Taqman探针进行实时PCR扩增,研究反应的特异性和检测限,并用所建立的方法对16份市售原料乳进行EPEC检测.结果表明,所用引物和探针可高效扩增出目的片段,与原料乳中其他常见致病菌无交叉反应,经2 h增菌后检测限为8.8 mL-1.对16份市售原料乳样品,检出2份含有EPEC,血清型分别为O111:K58(B4)和O127a:K63(B18).全部检测过程只需5h,可用于原料乳中EPEC的快速检测和污染调查.     

Pulsed field, PCR ribotyping and multiplex PCR analysis of Yersinia enterocolitica strains isolated from meat food in San Luis Argentina

The characterization of phenotypic and genotypic virulence markers of Yersinia enterocolitica strains belonging to biotypes (B) 1A, 2 and 3, mostly isolated from food in San Luis, Argentina, and the assessment of their genotypic diversity using PFGE and PCR ribotyping, were performed in our laboratory for the first time. Thirty five Y. enterocolitica strains, two reference strains and 33 strains isolated in our laboratory were studied. The presence of virF, ail, ystA, and myfA genes was investigated by multiplex PCR. The pathogenic potential of B1A strains, the most predominant biotype of Y. enterocolitica strains isolated from meat in our region, was investigated by simple PCR. Four B1A strains were positive for ystB gene. Four Y. enterocolitica 2/O:9 (bio/serotype) and two 3/O:5 strains isolated in our laboratory showed virulence-related results in the phenotypic tests and multiplex PCR. A good correlation between the expression of virulence markers and their corresponding genotypes was observed for most strains. Sixteen genomic types (GT) and 9 different intergenic spacer region (SR) groups were generated by PFGE and PCR ribotyping, respectively. In both cases the Y. enterocolitica 2/O:9 strains were separately clustered from 1A and 3/O:5 strains. Meat foods might be vehicles of transmission of pathogenic Y. enterocolitica strains in our region.