Rapid detection of Escherichia coli O157:H7 with multiplex real-time PCR assays

A SYBR Green LightCycler PCR assay using a single primer pair allowed simultaneous detection of stx1 and/or stx2 of Escherichia coli O157:H7. A distinct sequence of the Shiga-like toxin genes was amplified to yield products of 227 and/or 224 bp, respectively. The two products were distinguished by melting point curve analysis.     

Detection of Escherichia coli O157:H7 in soil and water using multiplex PCR

AIMS: To evaluate the suitability of a multiplex PCR-based assay for sensitive and rapid detection of Escherichia coli O157:H7 in soil and water. METHODS AND RESULTS: Soil and water samples were spiked with E. coli O157:H7 and subjected to two stages of enrichment prior to multiplex PCR. Detection sensitivities were as high as 1 cfu ml(-1) drinking water and 2 cfu g(-1) soil. Starvation of E. coli O157:H7 for 35 d prior to addition to soil did not affect the ability of the assay to detect initial cell numbers as low as 10 cfu g(-1) soil. Use of an 8-h primary enrichment enabled detection of as few as 6 cfu g(-1) soil, and 10(4) cfu g(-1) soil with a 6-h primary enrichment. When soil was inoculated with 10(5) cfu g(-1), the PCR assay indicated persistence of E. coli O157:H7 during a 35 d incubation. However, when soil was inoculated with lower numbers of pathogen, PCR amplification signals indicated survival to be dependent on cell concentration. CONCLUSIONS: A multiplex PCR-based assay, in combination with an enrichment strategy enabled sensitive and rapid detection of E. coli O157:H7 in soil and water. SIGNIFICANCE AND IMPACT OF THE STUDY: The ability to sensitively detect E.coli O157:H7 in environmental material within one working day represents a considerable advancement over alternative more time-consuming methods for detection of this pathogen.     

Simultaneous detection of Salmonella spp and Escherichia coli O157:H7 by multiplex PCR

Contamination of foods with pathogens such as Escherichia coli O157:H7 and Salmonella is a major concern worldwide and rapid, sensitive, and reliable methods are needed for detection of these organisms. Since these pathogens can contaminate similar foods and other types of samples, a multiplex polymerase chain reduction (PCR) was designed to allow simultaneous detection of both E. coli O157:H7 and Salmonella spp directly from enrichment cultures. Samples of apple cider, beef carcass wash water, ground beef, and bovine feces were inoculated with both E. coli O157:H7 and S. typhimurium at various bacterial levels. Following enrichment culturing for 20–24 h at 37°C in modified EC broth or buffered peptone water both containing novobiocin, the samples were subjected to a DNA extraction technique or to immunomagnetic separation then tested by the multiplex PCR assay. Four pairs of primers were employed in the PCR: primers for amplification of E. coli O157:H7 eaeA, stx _1/2 and plasmid sequences and for amplification of a portion of the Salmonella invA gene. Four fragments of the expected sizes were amplified in a single reaction and visualized following agarose gel electrophoresis in all the samples inoculated with ≤ 1 CFU g⁻¹ or ml⁻¹. Results can be obtained in approximately 30 h. The multiplex PCR is a potentially powerful technique for rapid and sensitive co-detection of both pathogens in foods and other types of samples. Received 28 December 1997/ Accepted in revised form 19 March 1998     

Comparison of an immunochromatographic method and the TaqMan E. coli O157:H7 assay for detection of Escherichia coli O157:H7 in alfalfa sprout spent irrigation water and in sprouts after blanching

An immunochromatographic-based assay (Quixtrade mark E. coli O157 Sprout Assay) and a polymerase chain reaction (PCR)-based assay (TaqMan E. coli O157:H7 Kit) were used to detect Escherichia coli O157:H7 strain 380-94 in spent irrigation water from alfalfa sprouts grown from artificially contaminated seeds. Ten, 25, 60, or 100 seeds contaminated by immersion for 15 min in a suspension of E. coli O157:H7 at concentrations of 10(6) or 10(8) cfu/ml were mixed with 20 g of non-inoculated seeds in plastic trays for sprouting. The seeds were sprayed with tap water for 15 s every hour and spent irrigation water was collected at intervals and tested. E. coli O157:H7 was detected in non-enriched water by both the TaqMan PCR (30 of 30 samples) and the immunoassay (9 of 24 samples) in water collected 30 h from the start of the sprouting process. However, enrichment of the spent irrigation water in brain heart infusion (BHI) broth at 37 degrees C for 20 h permitted detection of E. coli O157:H7 in water collected 8 h from the start of sprouting using both methods, even in trays containing as few as 10 inoculated seeds. The TaqMan PCR assay was more sensitive (more positive samples were observed earlier in the sprouting process) than the immunoassay; however, the immunoassay was easier to perform and was more rapid. At 72 h after the start of the sprouting process, the sprouts were heated at 100 degrees C for 30 s to determine the effectiveness of blanching for inactivation of E. coli O157:H7. All of the 32 samples tested with the TaqMan assay and 16 of 32 samples tested with the Quixtrade mark assay gave positive results for E. coli O157:H7 after enrichment of the blanched sprouts at 37 degrees C for 24 h. In addition, the organism was detected on Rainbow Agar O157 in 9 of 32 samples after 24 h of enrichment of the blanched sprouts. In conclusion, E. coli O157:H7 was detected in spent irrigation water collected from sprouts grown from artificially contaminated seeds by both the TaqMan and Quixtrade mark assays. The data also revealed that blanching may not be effective to completely inactivate all the E. coli O157:H7 that may be present in sprouts.     

Improved PCR assay for the specific detection and quantitation of Escherichia coli serotype O157 in water

Escherichia coli serotype O157 is still a major global healthcare problem. However, only limited information is now available on the molecular and serological detection of pathogenic bacteria. Therefore, the development of appropriate strategies for their rapid identification and monitoring is still needed. In general, the sequence analysis based on stx, slt, eae, hlyA, rfb, and fliC _h7 genes is widely employed for the identification of E. coli serotype O157; but there have been critical defects in the diagnosis and identification of E. coli serotype O157, in that they are also present in other E. coli serogroups. In this study, NCBI-BLAST searches using the nucleotide sequences of the putative regulatory protein gene from E. coli O157:H7 str. Sakai found sequence difference at the serotype level. The specific primers from the putative regulatory protein gene were designed and investigated for their sensitivity and specificity for detecting the pathogen in environment water samples. The specificity of the primer set was evaluated using genomic DNA from 8 isolates of E. coli serotype O157 and 32 other reference strains. In addition, the sensitivity and specificity of this assay were confirmed by successful identification of E. coli serotype O157 in environmental water samples. In conclusion, this study showed that the newly developed quantitative serotype-specific PCR method is a highly specific and efficient tool for the surveillance and rapid detection of high-risk E. coli serotype O157.     

RAPID detection and quantification of E. coli O157/O26/O111 in minced beef by real-time PCR

AIM: To develop a real-time PCR detection procedure for Escherichia coli O111, O26 and O157 from minced meat. METHODS AND RESULTS: Strains (n = 8) of each of E. coli O26, E. coli O111 and E. coli O157 were inoculated at ca 10-20 CFU g(-1) into minced retail meat and enriched for 6 h at 41.5 degrees C as follows: E. coli O26 in tryptone soya broth (TSB) supplemented with cefixime (50 microg l(-1)), vancomycin (40 mg l(-1)) and potassium tellurite (2.5 mg l(-1)); E. coli O111 in TSB supplemented with cefixime (50 microg l(-1)) and vancomycin (40 mg l(-1)); E. coli O157 in E. coli broth supplemented with novobiocin (20 mg l(-1)). DNA was extracted from the enriched cultures, and detected and quantified by real-time PCR using verotoxin (vt1 and vt2) and serogroup (O157 per gene; O26 fliC-fliA genes and O111 wzy gene) specific primers. CONCLUSIONS: The methods outlined were found to be sensitive and specific for the routine detection of E. coli O111, O26 and O157 in minced beef. SIGNIFICANCE AND IMPACT OF THE STUDY: The enrichment, isolation and detection procedures used in this study provide a rapid routine-based molecular method for the detection and differentiation of E. coli O26, O111 and O157 from minced meat.     

Characterization of rpoS alleles in Escherichia coli O157:H7 and in other E. coli serotypes

The rpoS nucleotide and predicted amino acid sequences from three Escherichia coli O157:H7 isolates were compared with those from three other E. coli isolates, including the likely O157:H7 progenitor, E. coli O55:H7. These clinical and environmental isolates all had identical sigma S amino acid sequences, while laboratory strains K12 and DH1 had three and one amino acid alterations, respectively, in comparison with the majority sequence. To extend the analysis of sigma S sequence conservation to include other Gram-negative bacteria, the E. coli sigma S sequences were compared with those from diverse Gram-negative organisms; sigma S sequence identities ranged from 50.2 to 99.7% among the available sequences. The results further confirm the existence of rpoS alleles among different E. coli strains, although all strains were classified as acid-resistant with survival rates > 10% after 2 h exposure to pH 2.5. It was also found that all E. coli O157:H7 isolates tested had a unique nucleotide at position 543, thus differentiating these strains from other E. coli serotypes.     

Rapid and sensitive detection of Escherichia coli O157:H7 in bovine faeces by a multiplex PCR

Cattle are considered the major reservoir for Escherichia coli O157:H7, one of the newly emerged foodborne human pathogens of animal origin and a leading cause of haemorrhagic colitis in humans. A sensitive test that can accurately and rapidly detect the organism in the food animal production environment is critically needed to monitor the emergence, transmission, and colonization of this pathogen in the animal reservoir. In this study, a novel multiplex polymerase chain reaction (PCR) assay was developed by using 5 sets of primers that specifically amplify segments of the eaeA, slt-I, slt-II, fliC, rfbE genes, which allowed simultaneous identification of serotype O157:H7 and its virulence factors in a single reaction. Analysis of 82 E. coli strains (49 O157:H7 and 33 non-O157:H7) demonstrated that this PCR system successfully distinguished serotype O157:H7 from other serotypes of E. coli and provided accurate profiling of the shiga-like toxins and the intimin adhesin in individual strains. This multiplex PCR assay did not cross-react with the background bacterial flora in bovine faeces and could detect a single O157:H7 organism per gram of faeces when combined with an enrichment step. Together, these results indicate that the multiplex PCR assay can be used for specific identification and profiling of E. coli O157:H7 isolates, and may be applied to rapid and sensitive detection of E. coli O157:H7 in bovine faeces when combined with an enrichment step.     

Multiplex PCR assay for the detection and quantification of Campylobacter spp., Escherichia coli O157:H7, and Salmonella serotypes in water samples

Three pathogens, Campylobacter, Salmonella, and Shiga-toxin-producing Escherichia coli, are leading causes of bacterial gastroenteritis in the United States and worldwide. Although these three bacteria are typically considered food-borne pathogens, outbreaks have been reported due to contaminated drinking water and irrigation water. The aim of this research was to develop two types of PCR assays that could detect and quantify three pathogens, Campylobacter spp., E. coli O157:H7, and Salmonella spp., in watershed samples. In conventional PCR, three target strains were detected by multiplex PCR (m-PCR) using each specific primer pair simultaneously. Under optimized m-PCR conditions, the assay produced a 90-bp product for Campylobacter jejuni, a 150-bp product for E. coli O157:H7, and a 262-bp product for Salmonella Typhimurium, and the limitation of detection was approximately 700 copies for all three bacteria. In addition, real-time PCR was performed to quantify the three pathogens using SYBR green fluorescence. The assay was designed so that each target had a different melting temperature [C. jejuni (80.1 °C), E. coli O157:H7 (83.3 °C), and S. Typhimurium (85.9 °C)]. Therefore, this system could quantify and distinguish three pathogens simultaneously in a single reaction.     

Direct PCR detection of Escherichia coli O157:H7

AIMS: This paper reports a simple, rapid approach for the detection of Shiga toxin (Stx)-producing Escherichia coli (STEC). METHODS AND RESULTS: Direct PCR (DPCR) obviates the need for the recovery of cells from the sample or DNA extraction prior to PCR. Primers specific for Stx-encoding genes stx1 and stx2 were used in DPCR for the detection of E. coli O157:H7 added to environmental water samples and milk. CONCLUSIONS: PCR reactions containing one cell yielded a DPCR product. SIGNIFICANCE AND IMPACT OF THE STUDY: This should provide an improved method to assess contamination of environmental and other samples by STEC and other pathogens.     

Multiplex PCR for detection of Escherichia coli O157:H7 in foods

Escherichia coli O157:H7 is well known enterohemorrhagic pathogen responsible for infections among animals including a man. The main source of this bacterium is cattle, that is mostly asymptomatic and through that E. coli O157:H7 can simple transfer to food products. Therefore, there is a need for rapid, sensitive and specific detection method. The present work is focused on its detection by a heptaplex polymerase chain reaction, which targets genes from known virulent regions of E. coli O157:H7. According to obtained results this approach is able to reach the detection sensitivity of 4 colony-forming units (CFU) from a culture and 6 and 8 CFU from milk and meat samples, respectively, independently of tested sample volume.     

Confirmation of viable E. coli O157:H7 by enrichment and PCR after rapid biosensor detection

Many rapid tests have been developed for the detection of Escherichia coli O157:H7 from complex matrices such as food and water. However, many of these methods rely on traditional culture steps for confirmation, which can take an extra 24-48 h. The fiber optic biosensor has been used to rapidly detect pathogens from complex matrices. In this paper, we demonstrate a method using a rapid biosensor assay, recovery through a short enrichment, and PCR to detect and confirm the presence of at least 10(3) CFU/ml of E. coli O157:H7 in a sample in less than 10 h.     

Rapid PCR confirmation of E. coli O157:H7 after evanescent wave fiber optic biosensor detection

Escherichia coli O157:H7 is an enteric pathogen of public health importance, which is monitored by several government agencies. Many rapid detection tests have been developed to identify foodstuff and water supplies contaminated by E. coli O157:H7. However, these methods can be time consuming (24-48 h) due to the need to culture the bacteria to confirm detection results. Fiber optic biosensors can rapidly detect pathogens from complex matrices, yet confirmation tests can take up to 10h to complete. In addition, fiber optic biosensors can also be used to reduce the impact of PCR inhibitors present in complex matrices such as food and water. This paper presents methodologies to reduce the time necessary for confirmation from 10 to about 2 h, by direct PCR of bacteria from the fiber optic waveguides without the need for culture or enrichment steps.     

致病性大肠埃希菌和沙门菌毒力岛基因的双重PCR检测方法的建立

目的实现对致病性大肠埃希菌（E．coli）、沙门菌（Salmonella）的同时检测,建立快速灵敏的双重PCR检测方法。方法以致病性大肠埃希菌和沙门菌毒力岛基因为研究对象,根据GenBank发表的大肠埃希菌和沙门菌毒力岛基因序列,分别设计合成了大肠埃希菌毒力岛irpl、irl）2和fyuA,沙门菌毒力岛mgtC、sseL和sopB等6对引物,以禽致病性大肠埃希菌（CVCC1565）菌株和沙门菌（ATCC9150）菌株的核酸混合物为模板,经引物特异性试验,引物组合,成功建立了快速鉴别检测致病性大肠埃希菌和沙门菌的双重PCR方法。结果特异性试验结果显示,引物irpl、irp2和fyuA仅能扩增出大肠埃希菌（CVCC1565）的特异性片段,大小分别是799、414和948bp;引物mgtC、sseL和sopB仅能扩增出沙门菌（ATCC9150）的特异性片段,大小分别是500、269和1000bp。敏感性试验结果表明大肠埃希菌和沙门菌的最低检测限分别为2．2×101CFU／mL和2．0×101CFU／mL。结论本研究建立的双重PCR方法具有特异性强、敏感性高、快速简便等特点,可用于致病性大肠埃希菌和沙门菌的联合检测与鉴别诊断。     

Comparison of multiplex PCR,and an immunochromatographic method sensitivity for the detection of Escherichia coli O157:H7 in minced beef

In this study, the sensitivities of multiplex PCR and an immuno-chromatographic methods to detect Escherichia coli O157:H7 in minced beef were compared. The detection of Escherichia coli O157:H7 in minced beef inoculated with 1-100 cells of this bacterium was possible after enrichment of culture and subsequent analysis by either of the two methods. Enrichment conditions were eight hours of incubation at 37 degrees C or 42 degrees C in a non-selective medium (Buffered Peptone Water). Multiplex PCR analysis was performed using three primer sets with analysis by gel electrophoresis. The Quix immuno-chromatographic assay which is a new kit being marketed by New Horizons Diagnostics, Columbia, MD, was used for immunological analysis of the enriched broths.The sensitivity of both tests was similar. The results depended on the concentration of the specific bacterium in the culture since the influence of the proportion of other bacteria to the E. coli O157:H7 was not observed. The data suggests that either method or used together, when coupled with an enrichment technique, could provide a rapid mean to detect the presence of this pathogen in minced meat samples.     

Flow-through immunofiltration assay system for rapid detection of E. coli O157:H7

A flow-through amperometric immunofiltration assay system based on disposable porous filter-membranes for rapid detection of Escherichia coli O157:H7 has been developed. The analytical system utilizes flow-through, immunofiltration and enzyme immunoassay techniques in conjunction with an amperometric sensor. The parameters affecting the immunoassay such as selection of appropriate filter membranes, membrane pore size, antibody binding capacity and the concentrations of immunoreagents were investigated and optimized. Non-specific adsorption of the enzyme conjugate was investigated and minimized. A sandwich scheme of immunoassay was employed and the immunofiltration system allows to specifically and directly detect E. coli cells with a lower detection limit of 100 cells/ml. The working range is from 100 to 600 cells/ml with an overall analysis time of 30 min. No pre-enrichment was needed. This immunosensor can be easily adapted for assay of other microorganisms and may be a basis for a new class of highly sensitive bioanalytical devices for rapid quantitative detection of bacteria.     

Detection of virulent Escherichia coli O157 strains using multiplex PCR and single base sequencing for SNP characterization

Aims: To compare 167 Norwegian human and nonhuman Escherichia coli O157:H7/NM (nonmotile) isolates with respect to an A/T single nucleotide polymorphism (SNP) in the tir gene and to detect specific SNPs that differentiate STEC O157 into distinct virulence clades (1–3 and 8). Methods and Results: We developed a multiplex PCR followed by single base sequencing for detection of the SNPs, and examined the association among SNP genotype, virulence profile (stx and eae status), multilocus variable number of tandem repeats analysis (MLVA) profile and clinical outcome. We found an over‐representation of the T allele among human strains compared to nonhuman strains, including 5/6 haemolytic‐uraemic syndrome cases. Fourteen strains belonged to clade 8, followed by two clade 2 strains. No clade 1 nor 3 isolates were observed. stx1 in combination with either stx2_EDL933 or stx2c were frequently observed among human strains, whereas stx2c was dominating in nonhuman strains. MLVA indicated that only single cases or small outbreaks with E. coli O157 have been observed in Norway through the years 1993–2008. Conclusion: We observed that the tir‐255 A/T SNP and the stx status were different between human and nonhuman O157 strains. No major outbreaks were observed, and only a few strains were differentiated into the virulence clades 2 and 8. Significance and Impact of the Study: The detection of virulence clade‐specific SNPs enables the rapid designation of virulent E. coli O157 strains, especially in outbreak situations.     

Development of primers to O-antigen biosynthesis genes for specific detection of Escherichia coli O157 by PCR.

The chemical composition of each O-antigen subunit in gram-negative bacteria is a reflection of the unique DNA sequences within each rfb operon. By characterizing DNA sequences contained with each rfb operon, a diagnostic serotype-specific probe to Escherichia coli O serotypes that are commonly associated with bacterial infections can be generated. Recently, from an E. coli O157:H7 cosmid library, O-antigen-positive cosmids were identified with O157-specific antisera. By using the cosmid DNAs as probes, several DNA fragments which were unique to E. coli O157 serotypes were identified by Southern analysis. Several of these DNA fragments were subcloned from O157-antigen-positive cosmids and served as DNA probes in Southern analysis. One DNA fragment within plasmid pDS306 which was specific for E. coli O157 serotypes was identified by Southern analysis. The DNA sequence for this plasmid revealed homology to two rfb genes, the first of which encodes a GDP-mannose dehydratase. These rfb genes were similar to O-antigen biosynthesis genes in Vibrio cholerae and Yersinia enterocolitica serotype O:8. An oligonucleotide primer pair was designed to amplify a 420-bp DNA fragment from E. coli O157 serotypes. The PCR test was specific for E. coli O157 serotypes. PCR detected as few as 10 cells with the O157-specific rfb oligonucleotide primers. Coupled with current enrichment protocols, O157 serotyping by PCR will provide a rapid, specific, and sensitive method for identifying E. coli O157.     

A high density microelectrode array biosensor for detection of E. coli O157:H7

A high density microelectrode array biosensor was developed for the detection of Escherichia coli O157:H7. The biosensor was fabricated from (100) silicon with a 2 microm layer of thermal oxide as an insulating layer, an active area of 9.6 mm2 and consists of an interdigitated gold electrode array. The sensor surface was functionalised for bacterial detection using heterobifunctional crosslinkers and immobilised polyclonal antibodies to create a biological sensing surface. Bacteria suspended in solution became attached to the immobilised antibodies when the biosensor was tested in liquid samples. The change in impedance caused by the bacteria was measured over a frequency range of 100 Hz-10 M Hz. The biosensor was evaluated for E. coli O157:H7 detection in pure culture and inoculated food samples. The biosensor was able to discriminate between cellular concentrations of 10(4)-10(7)CFU/mL and has applications in detecting pathogens in food samples.