Polymerase chain reaction assay for detection of Escherichia coli O157: H7 and Escherichia coli O157: H-.

The DNA band patterns generated by polymerase chain reaction (PCR) using the du2 primer and template DNAs from various strains of Escherichia coli and non-E. coli bacteria were compared. Among three to five prominent bands produced, the three bands at about 1.8, 2.7, and 5.0 kb were detected in all of the E. coli O157 strains tested. Some nonpathogenic E. coli and all pathogenic E. coli except E. coli O157 showed bands at 1.8 and 5.0 kb. It seems that the band at 2.7 kb is specific to E. coli O157. Sequence analysis of the 2.7-kb PCR product revealed the presence of a DNA sequence specific to E. coli O157:H- and E. coli O157:H7. Since the DNA sequence from base 15 to base 1,008 of the PCR product seems to be specific to E. coli O157, a PCR assay was carried out with various bacterial genomic DNAs and O157-FHC1 and O157-FHC2 primers that amplified the region between base 23 and base 994 of the 2.7-kb PCR product. A single band at 970 bp was clearly detected in all of the strains of E. coli O157:H- and E. coli O157:H7 tested. However, no band was amplified from template DNAs from other bacteria, including both nonpathogenic and pathogenic E. coli except E. coli O157. All raw meats inoculated with E. coli O157:H7 at 3 x 10(0) to 3.5 x 10(2) CFU/25 g were positive both for our PCR assay after cultivation in mEC-N broth at 42 degrees C for 18 h and for the conventional cultural method.     

Molecular beacon polymerase chain reaction detection of Escherichia coli O157:H7 in milk

A fluorescently labeled oligonucleotide probe (molecular beacon) was applied to detect Escherichia coli O157:H7 in artificially contaminated skim milk during polymerase chain reaction (PCR) amplification of extracted DNA. The probe was designed to hybridize with a region of the slt-II gene coding for the A subunit and to fluoresce when the hairpin-stem conformation was linearized upon hybridization to the target sequence. The molecular beacon was incorporated into PCR reactions containing DNA extracted from artificially contaminated skim milk. The degree of fluorescence was monitored in PCR reactions containing 10(3), 10(5), and 10(7) CFU of E. coli O157:H7 per ml and was found to correlate with the amount of template in each reaction. Fluorescence significantly increased above background levels by cycle 8, 14, or 14 in reactions containing DNA from the 10(7)-, 10(5)-, or 10(3)-CFU/ml template, respectively (P < 0.05). Molecular beacon PCR demonstrated positive results more rapidly than traditional agarose gel electrophoresis analysis of PCR products. Use of molecular beacons allows real-time monitoring of PCR reactions, and the closed-tube format allows simultaneous detection and confirmation of target amplicons without the need for agarose gel electrophoresis and/or Southern blotting. This is the first report of a stem-and-loop molecular beacon being applied for direct detection of a pathogen in food.     

Evaluation of methods to improve detection of Escherichia coli O157:H7 in fresh produce by multiplex polymerase chain reaction

Multiplex polymerase chain reaction (PCR) analysis was used to detect two genes encoding Shiga-like toxins (stx1 and stx2) and a universal Escherichia coli gene (gadA/B) in fresh produce spiked with E. coli O157:H7. Current U.S. Food and Drug Administration procedures for the analysis of fresh produce include the use of the rinsate from an initial rinse for the analysis of several potential pathogens, including E. coli O157:H7. In this study, several procedures were evaluated for their ability to increase the sensitivity of PCR analysis of rinsates from 15 types of produce. The procedures evaluated included the preliminary clarification and concentration of templates by centrifugation and the treatment of templates with compounds reported to facilitate nucleic acid amplification, including polyvinlypolypyrrolidone (PVPP), nonfat dry milk (NFDM), and InstaGene. The preliminary concentration of rinsates resulted in moderate improvements in detection sensitivity. The use of PVPP-treated templates in PCR reaction mixtures did not further improve sensitivity, but the inclusion of NFDM-treated templates increased sensitivity by an order of magnitude for 12 rinsates. The incorporation of InstaGene also improved the detection capability of the analysis; this procedure yielded the strongest gel bands for eight rinsates. However, for four other rinsates, the use of this reagent decreased sensitivity; these four rinsates were those for the produce varieties with the largest surface areas and were the most turbid rinsates. The use of facilitating compounds to block PCR inhibition may enable an analysis for Shiga toxin-producing E. coli in fresh produce to be completed in 1 to 2 days, rather than the 5 days required for current methods.     

Quantitative detection of Escherichia coli O157:H7 in ground beef by the polymerase chain reaction

A method for quantitative detection of Escherichia coli O157:H7 based on the polymerase chain reaction (PCR) was developed. The method used the NIH Image 1·61 software program to quantitatively analyse the intensity of the fluorescent image of the amplified PCR product. Based on the PCR with SLT1 and SLT2 primers used separately, a log-linear relationship between the numbers of cfu of E. coli O157:H7 inoculated into ground beef and the intensity of the PCR products was achieved with and without enrichment. Without enrichment, 150 cfu of E. coli O157:H7 per gram of ground beef were detected. In contrast, the detection limit decreased to 1·2 cfu g⁻¹ of ground beef using SLT1 and SLT2 primers after 4·5 h of enrichment using modified EC broth with 20 μg ml⁻¹ of novobiocin.     

基于实时荧光聚合酶链式反应快速检测食源性大肠埃希氏菌O157:H7

目的 建立一种快速、灵敏、特异、高效的食源性大肠埃希氏菌O157:H7型实时荧光聚合酶链式反应(polymerase chain reaction, PCR)检测方法。方法 针对大肠埃希氏菌O157:H7型的O抗原特异基因rfbE保守区域设计特异性引物和探针, 合成基因片段绘制标准曲线, 在菌液基因组DNA和质粒双层面调试优化以完成方法的初步建立。其后, 对该方法的特异性、敏感性、重复性等进行全面的质量评估验证。结果 该方法特异性100%; 基因组DNA检测敏感性为7.11×102 fg/μL; 纯培养物水平检测敏感性为1.0×102 CFU/mL; 重复性变异系数在0.10%~1.00%之间; 标准曲线相关系数r2为0.9994。结论 成功建立了一种性能良好的大肠埃希氏菌O157:H7型实时荧光探针PCR快速检测方法, 该方法具有灵敏度高、特异性强、扩增时间短, 仅为35 min的特点, 可用于疑似大肠埃希氏菌O157:H7型污染样品的快速诊断检测。     

Escherichia coli O157:H7 and its significance in foods

Escherichia coli O157:H7 was conclusively identified as a pathogen in 1982 following its association with two food-related outbreaks of an unusual gastrointestinal illness. The organism is now recognized as an important cause of foodborne disease, with outbreaks reported in the U.S.A., Canada, and the United Kingdom. Illness is generally quite severe, and can include three different syndromes, i.e., hemorrhagic colitis, hemolytic uremic syndrome, and thrombotic thrombocytopenic purpura. Most outbreaks have been associated with eating undercooked ground beef or, less frequently, drinking raw milk. Surveys of retail raw meats and poultry revealed E. coli O157:H7 in 1.5 to 3.5% of ground beef, pork, poultry, and lamb. Dairy cattle, especially young animals, have been identified as a reservoir. The organism is typical of most E. coli, but does possess distinguishing characteristics. For example, E. coli O157:H7 does not ferment sorbitol within 24 h, does not possess beta-glucuronidase activity, and does not grow well or at all at 44-45.5 degrees C. The organism has no unusual heat resistance; heating ground beef sufficiently to kill typical strains of salmonellae will also kill E. coli O157:H7. The mechanism of pathogenicity has not been fully elucidated, but clinical isolates produce one or more verotoxins which are believed to be important virulence factors. Little is known about the significance of pre-formed verotoxins in foods. The use of proper hygienic practices in handling foods of animal origin and proper heating of such foods before consumption are important control measures for the prevention of E. coli O157:H7 infections.     

Influence of growth in a food medium on the detection of Escherichia coli O157:H7 by polymerase chain reaction

The effects of storage time and growth in broth culture and in a food medium on the efficiency of Escherichia coli O157: H7 DNA extraction and on the sensitivity of polymerase chain reaction (PCR) detection of E. coli O157:H7 were investigated. Detection limits were evaluated with dilution series PCR targeting the slt-II gene. The relationship between cell density and DNA yield was generally log-linear for pure cultures of E coli O157:H7. When the bacteria were suspended in skim milk at a density of 10(6) CFU/ml. held at 4 degrees C, and sampled at 24-h intervals, cell density, total DNA yield, and PCR detection limits remained stable throughout the 96-h storage period. However, when E coli O157:H7 was grown in skim milk to a final cell density of 10(6) CFU/ml, PCR amplification efficiency was drastically reduced, although overall DNA yields from these samples were consistent with those for the samples in which E. coli O157:H7 growth was static over 96 h of storage at 4 degrees C. This result is most likely due to poor DNA purity, which was consistently observed when DNA was extracted from food matrices in which the pathogen was grown rather than stored. The results of this investigation underscore the likelihood that multiple components may drastically affect DNA extraction and PCR amplification efficiency in the detection of pathogens in the food matrix. It is clear that before nucleic acid amplification technologies are widely applied to food systems, it would be prudent to test their efficacy in multiple food matrices and under conditions in which the bacterial population is both static and actively growing.     

Poly-l-lysine-functionalized magnetic beads combined with polymerase chain reaction for the detection of Staphylococcus aureus and Escherichia coli O157:H7 in milk

Rapid and credible detection of pathogens is essential to prevent and control outbreaks of foodborne diseases. In this study, a poly-l-lysine-functionalized magnetic beads (PLL-MB) strategy combined with a PCR assay was established to detect Staphylococcus aureus. We also detected Escherichia coli O157:H7 to further verify the strategy for gram-negative bacteria detection. Poly-l-lysine has strong positive charges because of its amino groups, which can conjugate with the carboxyl of carboxyl magnetic beads. Furthermore, it can be used to combine with bacteria through electrostatic adsorption. Under optimum conditions, the developed PLL-MB complexes showed 90% capture efficiency in phosphate-buffered saline and 85% capture efficiency in milk for S. aureus detection. The limit of detection of the PLL-MB-PCR assay was 10² cfu/mL (1.8 × 10² cfu/mL for S. aureus and 7 × 10² cfu/mL for E. coli O157:H7) in phosphate-buffered saline and milk samples. The whole assay can be performed within 4 h. The proposed strategy showed a lower limit of detection when compared with the conventional PCR assay without enrichment. In addition, this method exhibited the advantages of a high-efficient, cost-efficient, and simple operation, indicating its potential applications in foodborne pathogen detection.     

A rapid detection of Escherichia coli O157:H7 by competition visual antigen macroarray

Pathogenic bacterial contamination is a serious problem for the food industry and in public health. Rapid, accurate and affordable testing for pathogenic bacterial strains is desirable. In this study, a competition visual antigen macroarray (CVAM) for rapid detection of Escherichia coli O157:H7 (E. coli O157:H7) has been developed. This array was able to utilize an HRP‐labeled anti‐E. coli O157:H7 MAb at a concentration of 1:20000 while having a similar sensitivity of 10 ⁵ CFU/ml for E. coli O157:H7 detection as double antibody sandwich array and conventional ELISA. The detection time of CVAM was short as 2 h and can be examined directly by the naked eye. Moreover, there was no cross‐reactivity in the detection of tested in this study. Application of CVAM for the detection of E. coli O157:H7 artificially contaminated milk samples was feasible that showed the antigen array could be applied in the detection of food pathogen infections.     

Detection and quantitation of enterohemorrhagic Escherichia coli O157, O111, and O26 in beef and bovine feces by real-time polymerase chain reaction

Enterohemorrhagic Escherichia coli (EHEC) O157:H7 and certain non-O157 EHEC serotypes (such as O26:H11, O26: NM, O11:H8, and O111:NM) have emerged as significant causes of human disease throughout the world. Important virulence attributes of EHEC are the intimin protein (encoded by the eae gene) and Shiga toxins 1 and 2 (encoded by the stx1 and stx2 genes, respectively). Two sets of real-time polymerase chain reaction (R-PCR) assays were developed for the simultaneous detection and quantitation of EHEC through the monitoring of the presence of the eae and stx genes, and these assays were evaluated. In the eaeR-PCR assay, three sets of primers and TaqMan probes were designed for the amplification and real-time detection of a portion of the eae gene specific to the EHEC O26, O111, and O157 serotypes. In the stxR-PCR assay, two sets of primers and TaqMan probes were used to amplify and detect the stx1 and stx2 genes. DNA prepared from 67 bacterial strains carrying known virulence markers was tested to determine the specificities of the two assays. In the eaeR-PCR assay, eaeO157- and eaeO111-specific primer-probe sets identified only EHEC O157 and O111 strains, respectively. The eaeO26-specific primer-probe set identified all EHEC 026 isolates and some Shiga toxin-negative serotypes of enteropathogenic E. coli and rabbit diarrheagenic E. coli. The stxR-PCR assay was able to identify only those strains carrying either or both of the Shiga toxin-encoding genes. The detection range of both R-PCR assays was linear over DNA concentrations corresponding to 10(3) to 10(8) CFU/ml of an EHEC strain. Both assays were able to detect and quantify very low levels (1 to 10 CFU/g of food or feces) of EHEC in feces and ground beef enriched for 16 h in a modified Trypticase soy broth. In conclusion, eae- and stx-based R-PCR assays are reliable and sensitive methods for the rapid screening and specific and quantitative detection of important serotypes of EHEC in cattle and in foods of bovine origin.     

Survival of Escherichia coli O157:H7 in bovine feces over time under various temperature conditions

Although Escherichia coli O157:H7 prevalence estimates in cattle have increased over time due to improvements in detection methods, fecal sample transport conditions from farm to microbiological laboratories for further analysis may be a factor for prevalence underestimation. The objective of this study was to compare and determine the survival characteristics of E. coli O157:H7 in bovine feces under various storage conditions that could be encountered during transport. Fecal pats were inoculated with a four-strain cocktail of antibiotic-resistant E. coli O157:H7 to contain approximately 1 x 10(5) CFU/g. Inoculated and control samples were taken after 0, 24, 48, 120, and 168 h at each storage temperature and examined for presence and numbers of E. coli O157:H7. Each sample was subdivided and placed at each of the four following temperatures: 37, 23, 4.4 degrees C, and in a plastic cooler with refrigerant packs (0, 4, 4, 21, and 23 degrees C at five sampling times, respectively) to simulate transportation conditions. A statistically significant decrease in the population of the pathogen was observed after 48 h in samples held at 37 degrees C (P < 0.01) and after 168 h at 4.4 degrees C (P = 0.02). At 37 degrees C, E. coli O157:H7 was not detected after 48 h, either by direct plating (P < 0.01) or by immunomagnetic separation. Overall, the results of this study showed that E. coli O157:H7 survived without significant detriment in bovine fecal material inside the cooler for up to 168 h. These results indicate that shipment and storage under these conditions before microbiological analysis would be acceptable and should not affect pathogen detection.     

食品中大肠杆菌O157:H7的几种检测方法的比较

目的评估平板分离培养法、免疫磁珠分离(IMS)法、VIDAS全自动酶标免疫测试系统、BAX全自动病原菌检测系统及环介导等温扩增(LAMP)技术在食品中检验肠出血型大肠埃希菌O157∶H7的特异性、敏感性。方法使用平板分离培养法、免疫磁珠分离法、VIDAS法、BAX法及LAMP法对人工制备的染菌猪肉样本进行检测,并对这几种方法进行比较。结果 BAX法和LAMP法的检出率最高,分别是89.1%和85.9%,免疫磁珠法和VIDAS法检出率次之,分别是75.0%和78.1%,传统分离培养法为43.8%。结论 BAX法和LAMP法具有快速、高效、特异性好、敏感性高的特点,可快速筛选食品中可能存在的肠出血型大肠埃希菌O157∶H7。     

Isolation of Escherichia coli O157:H7 from foods in Greece

The presence of Escherichia coli O157:H7 in various foods of animal origin was surveyed in northwestern Greece. Six hundred samples of unpasteurized cows', ewes' and goats' milk, raw minced meat, uncooked frozen beef hamburgers, sandwiches (containing ham or turkey, mixed vegetable salad with mayonnaise and lettuce), fresh traditional Greek pork sausages and swine intestines appropriate for traditional Greek kokoretsi were assayed for E. coli serogroup O157:H7 using the standard cultural method and the immunomagnetic separation technique. The pathogen was detected in 1 out of 100 (1.0%) samples of ewes' milk, 1 out of 75 (1.3%) fresh sausages and 1 out of 50 (2.0%) swine intestines prepared for kokoretsi. The isolated strains were nonsorbitol fermenters, MUG-negative, O157 agglutinating, verotoxin-producing and carried both VT1 and VT2 genes. The three isolated strains were tested for antibiotic resistance and were found to be susceptible to eight antimicrobial agents (ampicillin, chloramphenicol, kanamycin, nalidixic acid, norfloxacin, streptomycin, sulfamethoxazole-trimethoprim and tetracycline).     

Seasonal prevalence of Escherichia coli O157:H7 in beef cattle feces

Cattle are an asymptomatic reservoir of Escherichia coli O157:H7, but the bacterial colonization and shedding patterns are poorly understood. The prevalence and shedding of this human pathogen have been reported to be seasonal with rates typically increasing during warm months. The objectives of this study were (i) to assess the prevalence of E. coli O157:H7 in feces of feedlot cattle in Kansas during summer, fall, and winter months, and (ii) to characterize E. coli O157:H7 by screening for virulence factors. Of 891 fecal samples collected, 82 (9.2%) were positive for E. coli O157:H7. No significant differences in prevalence were detected among summer, fall, and winter months. The highest monthly prevalence (18.1%) was detected in February. All tested isolates were positive for stx2 (Shiga toxin 2) and eaeA (intimin) genes; 14 isolates (12.8%) also carried stx1. Our results indicate the prevalence of E. coli O157:H7 in beef cattle feces is not necessarily season dependent.     

Modifications to methods for the enumeration and detection of injured Escherichia coli O157:H7 in foods

Reliable methods are required for the detection and enumeration of potentially injured E. coli O157 in food in the presence of outnumbering competing bacteria. Selective agents can prevent or inhibit the recovery and subsequent multiplication of injured cells and direct inoculation, either into selective enrichment broths or onto selective agar plates is still used in many methods for E. coli O157 detection and enumeration. When compared with tryptone soya agar (TSA), sorbitol MacConkey agar (SMAC) was shown to underestimate the concentration of viable E. coli O157:H7 subjected to low pH and high NaCl concentration. Using a resuscitation stage on TSA followed by membrane transfer to SMAC improved recovery to levels obtained on TSA. The membrane method was used to monitor the numbers of artificially contaminated E. coli O157:H7 during the fermentation of a meat product and demonstrated better survival when compared to counts on SMAC. Six rapid methods for the detection of E. coli O157 in food (BAX E. coli O157, Reveal 8 E. coli O157-H7 screening test, VIP EHEC, VIDAS E. coli O157 (ECO), EHEC-Tek and Tecra E. coli O157 visual immunoassay), were evaluated using beetburgers, parsley and fermented meat artificially contaminated with injured cells. Methods using direct selective enrichment, with or without an elevated incubation temperature gave false-negative results. The incorporation of a non-selective pre-enrichment medium improved the detection rates of these assays by up to ten fold.     

Evaluation of the Reveal and SafePath rapid Escherichia coli O157 detection tests for use on bovine feces and carcasses

The Reveal (Neogen Corp., Lansing, Mich.) and SafePath (SafePath Laboratories LLC, St. Paul, Minn.) tests were evaluated for their performance as beef fecal and beef carcass Escherichia coli O157:H7 monitoring tests. Agreement between these tests and a reference test system was determined using naturally contaminated bovine feces and beef carcasses. The reference system utilized immunomagnetic separation with plating onto cefixime, tellurite, sorbitol MacConkey agar, followed by colony testing using a serum agglutination test for the O157 antigen. Relative to this reference method, the Reveal test showed a sensitivity of 46% and a specificity of 82% on bovine feces and a specificity of 99% on carcass samples. The SafePath test, demonstrated a significantly higher sensitivity at 79% and a similar specificity of 79%. On carcass samples the SafePath test performed similarly to the Reveal test, demonstrating a specificity of 100% relative to the reference system. There was an insufficient number of E. coli O157-positive carcass samples to estimate precisely the sensitivity of these two methods. Both methods show promise as rapid carcass monitoring tests, but further field testing to estimate sensitivity is needed to complete their evaluation. The proportion of positive fecal samples for E. coli O157:H7 by the reference method ranged from 10.2% to 36% in 10 lots of cattle with an overall mean of 17.3% (39/225). Quarter carcass sponging of 125 carcasses revealed 1.6% positive for the pathogen (2/125).     

Modified immunoliposome sandwich assay for the detection of Escherichia coli O157:H7 in apple cider

Detection of Escherichia coli O157:H7 in fruit juices such as apple cider is necessary for diagnosis of infection and epidemiological investigations. However, inhibitors in the apple cider, such as endogenous polyphenols and acids, often decrease the sensitivity of PCR assays and immunoassays, thus routinely requiring laborious cell separation steps to increase the sensitivity. In the current study, polyethylene glycol (PEG)-derivatized liposomes encapsulating sulforhodamine B were tagged with anti-E. coli O157:H7 antibodies and used in an immunoliposome sandwich assay for the detection of E. coli O157:H7 in apple cider. Even without prior separation, this assay can detect E. coli O157:H7 in apple cider samples inoculated with as few as 1 CFU/ml after an 8-h enrichment period. The lower limit of detection in pure cultures without enrichment was 7 x 10(3) CFU/ml (280 CFU/40-microl sample). PEGylated immunoliposomes are suitable as an analytical reagent for the detection of E. coli O157:H7 in fruit juices containing polyphenols.     

Escherichia coli O157:H7 genetic diversity in bovine fecal samples

Escherichia coli O157:H7 causes foodborne illness in humans; cattle are considered a primary reservoir for the organism, and transmission is often through contaminated food products or water. The objective of this study was to determine the genetic diversity of E. coli O157:H7 within a single individual bovine fecal sample based on pulsed-field gel electrophoresis (PFGE) typing. Fecal samples (n=601) were collected from dairy and beef cattle at three separate facilities, and E. coli O157:H7 was isolated by enrichment, immunomagnetic separation, and plating on selective medium. The prevalence of E. coli O157:H7 was 46 (7.7%) of 601. From each positive fecal sample, up to 10 putative colonies were tested, and isolates from samples with at least seven positive colonies were subtyped using PFGE and tested for six major virulence genes by multiplex PCR. A total of 254 E. coli O157:H7 isolates from 27 samples met these criteria and were included in PFGE analysis. Fifteen PFGE subtypes (<100% Dice similarity) were detected among the 254 isolates, and there were no common subtypes between the three locations. Seven (26%) of 27 fecal samples had E. coli O157:H7 isolates with different PFGE subtypes (mean=2.1) within the same sample. The virulence gene profiles of different isolates from the same sample were always identical, regardless of the number of PFGE types. The results of this study suggest that determining the PFGE pattern of a single isolate from a bovine sample may not be sufficient when comparing isolates from feces, hides, or carcasses, because multiple PFGE subtypes are present.     

Nanozyme-based lateral flow assay for the sensitive detection of Escherichia coli O157:H7 in milk

Lateral flow assay (LFA) has been applied in many fields due to its relative ease of use and cost-effectiveness. However, it has low sensitivity and its applications are limited. Probe materials play a significant role in improving the detection efficiency and sensitivity of LFA. In this study, by using concave palladium-platinum (Pd-Pt) nanoparticles as a nanozyme probe, we developed a sensitive LFA based on the sandwich format for qualitative and quantitative detection of Escherichia coli O157:H7. The sensitivity of the LFA was improved by applying the 3,3′,5,5′-tetramethylbenzidine (TMB) substrate onto the test line where the nanozyme was accumulated in the presence of analytes. The nanozyme showed high catalytic performance toward TMB and greatly enhanced the signal intensity of the test line. The sensitivity of the nanozyme-based LFA was 9.0 × 10² cfu/mL in milk, which was 111-fold higher than that of traditional colloidal gold-based LFA. The proposed method has remarkable potential in the detection of various pathogens in real samples.     

食品中大肠杆菌O157:H7的预测模型及风险评估

大肠杆菌O157:H7是严重危害人类健康的肠道致病菌,感染剂量极低,低达10个菌体。由该菌引起的食物中毒十分凶险,引起世界各国的重视。该菌引起的感染主要通过食品传播,因此加强食品卫生管理,防止食源性感染和流行尤为迫切。本文就大肠杆菌O157:H7在食品中的预测模型以及风险评估进行了概述。