scholarly journals Quantitative detection of E. coli, E. coli O157 and other shiga toxin producing E. coli in water samples using a culture method combined with real-time PCR

2006 ◽  
Vol 4 (4) ◽  
pp. 487-498 ◽  
Author(s):  
Leo Heijnen ◽  
Gertjan Medema
Keyword(s):  
Surface Water ◽  
Real Time ◽  
Shiga Toxin ◽  
Water Samples ◽  
Real Time Pcr ◽  
Matrix Effects ◽  
Quantitative Detection ◽  
Attractive Alternative ◽  
E Coli ◽  
Wastewater Samples

Recent water related outbreaks of shiga toxin producing E. coli O157 have resulted in increased attention of the water industry to this potentially deadly pathogen. Current methods to detect E. coli O157 and its virulence genes are laborious and time-consuming. Specificity, sensitivity and simple use of a real-time PCR method makes it an attractive alternative for the detection of STEC E. coli O157. This study describes the development and application of real-time PCR methods for the detection of E. coli O157, shiga toxin genes (Stx1 and Stx2) and E. coli. The specificity of the methods was confirmed by performing colony-PCR assays on characterized bacterial isolates, demonstrating the applicability of these assays as rapid tests to confirm the presence of E. coli or E. coli O157 colonies on culture plates. Sensitive culture-PCR methods were developed by combining culture enrichment with real-time PCR detection. This rapid method allowed detection of low concentrations of E. coli O157 in the presence of high concentrations of non-O157-E. coli (1:104). Culture-PCR methods were applied to 27 surface water and 4 wastewater samples. E. coli O157 and both Stx genes were detected in two wastewater samples, whereas only E. coli O157 was detected in two surface water samples. Culture-PCR methods were not influenced by matrix effects and also enabled quantitative (MPN) detection of E. coli in these samples.

Digoxigenin-Labeled Probe-Based Colony Blotting Assay for Rapid Quantification of Salmonella Serovars in Seafood and Water

2012 ◽  
Vol 95 (6) ◽  
pp. 1652-1655 ◽  
Author(s):  
Rakesh Kumar ◽  
K V Lalitha
Keyword(s):  
Real Time ◽  
Water Samples ◽  
Real Time Pcr ◽  
Quantitative Detection ◽  
Gene Probe ◽  
Pcr Assay ◽  
Nylon Membrane ◽  
Rapid Enumeration ◽  
Salmonella Serovars ◽  
Labeled Probe

Abstract A non-radio-labeled probe-based detection method was developed for rapid enumeration of Salmonella in seafood and water samples. A Salmonella-specific invA gene probe was developed using a digoxigenin-based non-radio labeling assay, which was evaluated with naturally contaminated seafood and water samples. The probe-based technique was further compared with the quantitative PCR assay. The method was specific for detection of different Salmonella serovars without any nonspecific hybridization with other Salmonella-related Enterobacteriaceae. The optimum labeling efficiency was determined for the labeled probe, and 10 pg/μL probe concentration was observed to be most efficient for detection of Salmonella colonies on nylon membrane. Quantification of Salmonella in naturally contaminated seafood and water samples (n = 21) was in the range 10–102 CFU/mL. The assay successfully quantified Salmonella in spiked seafood and water samples in the presence of background flora, and the entire assay was completed within 48 h. The probe-based assay was further evaluated with real-time PCR, and results showed that the assay was comparable to real-time PCR assay. Thus, this probe-based assay can be a rapid, useful, and alternative technique for quantitative detection of Salmonella in food, feed, and water samples.

Comparison of a New Enrichment Procedure for Shiga Toxin–Producing Escherichia coli with Five Standard Methods

2005 ◽  
Vol 68 (8) ◽  
pp. 1593-1599 ◽  
Author(s):  
MICHAEL A. GRANT
Keyword(s):  
Escherichia Coli ◽  
Real Time ◽  
Shiga Toxin ◽  
Real Time Pcr ◽  
Target Cells ◽  
Macconkey Agar ◽  
Standard Methods ◽  
E Coli ◽  
Canadian Health ◽  
Health Products

A new procedure for enrichment of Escherichia coli O157:H7 and other Shiga toxin–producing E. coli was compared to five standard methods: the British Public Health Laboratory Service, International Standard Method, U.S. Department of Agriculture, Canadian Health Products and Food Branch, and U.S. Food and Drug Administration. The new procedure was comparable to the standard methods in its ability to detect target cells inoculated into foods at approximately 1 CFU g−1. Comparisons were also made of the ability of the six enrichment procedures to detect E. coli O157:H7 against a large background of competitor microorganisms. In these experiments the new procedure yielded more target cells than the other five enrichments by two to three orders of magnitude as determined by enumeration on sorbitol MacConkey agar with tellurite and cefixime and Rainbow agar with tellurite and novobiocin and by verification of presumptive colonies by real-time PCR. For example, the population of enterohemorrhagic E. coli strain 6341 recovered on sorbitol MacConkey agar with tellurite and cefixime after enrichment with the experimental method was 2.42 × 108 CFU ml−1 and 1.80 × 106 CFU ml−1 after enrichment with the Canadian Health Products and Food Branch method, the second most effective in this experiment. In addition, broth cultures resulting from each of the six enrichment procedures were used to prepare templates for real-time PCR detection of stx1/stx2. Resulting threshold cycle (Ct) values after the experimental enrichment were similar to positive control values, whereas the five standard methods produced delayed Ct values or were not detected.

scholarly journals Quantitative Detection of Legionella pneumophila in Water Samples by Immunomagnetic Purification and Real-Time PCR Amplification of the dotA Gene

2005 ◽  
Vol 71 (7) ◽  
pp. 3433-3441 ◽  
Author(s):  
M. A. Yáñez ◽  
C. Carrasco-Serrano ◽  
V. M. Barberá ◽  
V. Catalán
Keyword(s):  
Cell Culture ◽  
Real Time ◽  
Legionella Pneumophila ◽  
Water Samples ◽  
Real Time Pcr ◽  
Limit Of Detection ◽  
Immunomagnetic Separation ◽  
Pcr Analysis ◽  
Quantitative Detection ◽  
Specific Sequence

ABSTRACT A new real-time PCR assay was developed and validated in combination with an immunomagnetic separation system for the quantitative determination of Legionella pneumophila in water samples. Primers that amplify simultaneously an 80-bp fragment of the dotA gene from L. pneumophila and a recombinant fragment including a specific sequence of the gyrB gene from Aeromonas hydrophila, added as an internal positive control, were used. The specificity, limit of detection, limit of quantification, repetitivity, reproducibility, and accuracy of the method were calculated, and the values obtained confirmed the applicability of the method for the quantitative detection of L. pneumophila. Moreover, the efficiency of immunomagnetic separation in the recovery of L. pneumophila from different kinds of water was evaluated. The recovery rates decreased as the water contamination increased (ranging from 59.9% for distilled water to 36% for cooling tower water), and the reproducibility also decreased in parallel to water complexity. The feasibility of the method was evaluated by cell culture and real-time PCR analysis of 60 samples in parallel. All the samples found to be positive by cell culture were also positive by real-time PCR, while only eight samples were found to be positive only by PCR. Finally, the correlation of both methods showed that the number of cells calculated by PCR was 20-fold higher than the culture values. In conclusion, the real-time PCR method combined with immunomagnetic separation provides a sensitive, specific, and accurate method for the rapid quantification of L. pneumophila in water samples. However, the recovery efficiency of immunomagnetic separation should be considered in complex samples.

Rapid and specific detection of Salmonella in water samples using real-time PCR and High Resolution Melt (HRM) curve analysis

2011 ◽  
Vol 64 (12) ◽  
pp. 2453-2459 ◽  
Author(s):  
G. N. van Blerk ◽  
L. Leibach ◽  
A. Mabunda ◽  
A. Chapman ◽  
D. Louw
Keyword(s):  
Surface Water ◽  
High Resolution ◽  
Real Time ◽  
Water Samples ◽  
Real Time Pcr ◽  
Curve Analysis ◽  
Pcr Assay ◽  
High Resolution Melt ◽  
Gene Target ◽  
Enrichment Step

A real-time PCR assay combined with a pre-enrichment step for the specific and rapid detection of Salmonella in water samples is described. Following amplification of the invA gene target, High Resolution Melt (HRM) curve analysis was used to discriminate between products formed and to positively identify invA amplification. The real-time PCR assay was evaluated for specificity and sensitivity. The assay displayed 100% specificity for Salmonella and combined with a 16–18 h non-selective pre-enrichment step, the assay proved to be highly sensitive with a detection limit of 1.0 CFU/ml for surface water samples. The detection assay also demonstrated a high intra-run and inter-run repeatability with very little variation in invA amplicon melting temperature. When applied to water samples received routinely by the laboratory, the assay showed the presence of Salmonella in particularly surface water and treated effluent samples. Using the HRM based assay, the time required for Salmonella detection was drastically shortened to less than 24 h compared to several days when using standard culturing methods. This assay provides a useful tool for routine water quality monitoring as well as for quick screening during disease outbreaks.

scholarly journals Detection of Shiga Toxin-ProducingEscherichia coliSerotypes O26:H11, O103:H2, O111:H8, O145:H28, and O157:H7 in Raw-Milk Cheeses by Using Multiplex Real-Time PCR

2011 ◽  
Vol 77 (6) ◽  
pp. 2035-2041 ◽  
Author(s):  
Jordan Madic ◽  
Noémie Vingadassalon ◽  
Carine Peytavin de Garam ◽  
Muriel Marault ◽  
Flemming Scheutz ◽  
...  
Keyword(s):  
Real Time ◽  
Genetic Markers ◽  
Shiga Toxin ◽  
Real Time Pcr ◽  
Raw Milk ◽  
Immunomagnetic Separation ◽  
Genetic Profile ◽  
High Genetic Diversity ◽  
E Coli ◽  
Three Samples

ABSTRACTShiga toxin (Stx)-producingEscherichia coli(STEC) strains are a diverse group of food-borne pathogens with various levels of virulence for humans. In this study, we describe the use of a combination of multiple real-time PCR assays for the screening of 400 raw-milk cheeses for the five main pathogenic STEC serotypes (O26:H11, O103:H2, O111:H8, O145:H28, and O157:H7). The prevalences of samples positive forstx, intimin-encoding gene (eae), and at least one of the five O group genetic markers were 29.8%, 37.3%, and 55.3%, respectively. The H2, H7, H8, H11, and H28fliCalleles were highly prevalent and could not be used as reliable targets for screening. Combinations ofstx,eaevariants, and O genetic markers, which are typical of the five targeted STEC serotypes, were detected by real-time PCR in 6.5% of the cheeses (26 samples) and includedstx-wzxO26-eae-β1(4.8%; 19 samples),stx-wzxO103-eae-ε (1.3%; five samples),stx-ihp1O145-eae-γ1(0.8%; three samples), andstx-rfbEO157-eae-γ1(0.3%; one sample). Twenty-eight immunomagnetic separation (IMS) assays performed on samples positive for these combinations allowed the recovery of seveneaeβ1-positive STEC O26:H11 isolates, whereas no STEC O103:H2, O145:H28, or O157:H7 strains could be isolated. Threestx-negative andeaeβ1-positiveE. coliO26:[H11] strains were also isolated from cheeses by IMS. Colony hybridization allowed us to recover STEC fromstx-positive samples for 15 out of 45 assays performed, highlighting the difficulties encountered in STEC isolation from dairy products. The STEC O26:H11 isolates shared the same virulence genetic profile as enterohemorrhagicE. coli(EHEC) O26:H11, i.e., they carried the virulence-associated genes EHEC-hlyA,katP, andespP, as well as genomic O islands 71 and 122. Except for one strain, they all contained thestx1variant only, which was reported to be less frequently associated with human cases thanstx2. Pulsed-field gel electrophoresis (PFGE) analysis showed that they displayed high genetic diversity; none of them had patterns identical to those of human O26:H11 strains investigated here.

A duplex real-time PCR assay for the quantitative detection of Naegleria fowleri in water samples

2007 ◽  
Vol 41 (1) ◽  
pp. 118-126 ◽  
Author(s):  
Jonas Behets ◽  
Priscilla Declerck ◽  
Yasmine Delaedt ◽  
Lieve Verelst ◽  
Frans Ollevier
Keyword(s):  
Real Time ◽  
Water Samples ◽  
Real Time Pcr ◽  
Quantitative Detection ◽  
Naegleria Fowleri ◽  
Pcr Assay ◽  
Nægleria Fowleri

Quantitative detection of human enteric adenoviruses in river water by microfluidic digital polymerase chain reaction

2014 ◽  
Vol 70 (3) ◽  
pp. 555-560 ◽  
Author(s):  
Naohiro Kishida ◽  
Naohiro Noda ◽  
Eiji Haramoto ◽  
Mamoru Kawaharasaki ◽  
Michihiro Akiba ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Real Time ◽  
River Water ◽  
Water Samples ◽  
Real Time Pcr ◽  
Quantitative Detection ◽  
Chain Reaction ◽  
Polymerase Chain ◽  
Digital Polymerase Chain Reaction ◽  
Enteric Adenoviruses

We describe an assay for simple and accurate quantification of human enteric adenoviruses (EAdVs) in water samples using a recently developed quantification method named microfluidic digital polymerase chain reaction (dPCR). The assay is based on automatic distribution of reaction mixture into a large number of nanolitre-volume reaction chambers and absolute copy number quantification from the number of chambers containing amplification products on the basis of Poisson statistics. This assay allows absolute quantification of target genes without the use of standard DNA. Concentrations of EAdVs in Japanese river water samples were successfully quantified by the developed dPCR assay. The EAdVs were detected in seven of the 10 samples (1 L each), and the concentration ranged from 420 to 2,700 copies/L. The quantified values closely resemble those by most probable number (MPN)-PCR and real-time PCR when standard DNA was validated by dPCR whereas they varied substantially when the standard was not validated. Accuracy and sensitivity of the dPCR was higher than those of real-time PCR and MPN-PCR. To our knowledge, this is the first study that has successfully quantified enteric viruses in river water using dPCR. This method will contribute to better understanding of existence of viruses in water.

Recirculating Immunomagnetic Separation and Optimal Enrichment Conditions for Enhanced Detection and Recovery of Low Levels of Escherichia coli O157:H7 from Fresh Leafy Produce and Surface Water

2007 ◽  
Vol 70 (12) ◽  
pp. 2717-2724 ◽  
Author(s):  
SUNEE HIMATHONGKHAM ◽  
MARY LEE DODD ◽  
JENNY K. YEE ◽  
DAVID K. LAU ◽  
RAYMOND G. BRYANT ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Surface Water ◽  
Real Time ◽  
Real Time Pcr ◽  
Immunomagnetic Separation ◽  
Escherichia Coli O157 ◽  
Simple Method ◽  
E Coli ◽  
Low Levels ◽  
Spinach Leaves

The objective of this study was to develop a rapid, simple method for enhanced detection and isolation of low levels of Escherichia coli O157:H7 from leafy produce and surface water using recirculating immunomagnetic separation (RIMS) coupled with real-time PCR and a standard culture method. The optimal enrichment conditions for the method also were determined. Analysis of real-time PCR data (CT values) suggested that incubation of lettuce and spinach leaves rather than rinsates provides better enrichment of E. coli O157:H7. Enrichment of lettuce or spinach leaves at 42°C for 5 h provided better detection than enrichment at 37°C. Extended incubation of surface water for 20 h at 42°C did not improve the detection. The optimized enrichment conditions were also employed with modified Moore swabs, which were used to sample flowing water sites. Positive isolation rates and real-time PCR results indicated an increased recovery of E. coli O157:H7 from all samples following the application of RIMS. Under these conditions, the method provided detection and/or isolation of E. coli O157:H7 at levels as low as 0.07 CFU/g of lettuce, 0.1 CFU/g of spinach, 6 CFU/100 ml of surface water, and 9 CFU per modified Moore swab. During a 6-month field study, modified Moore swabs yielded high isolation rates when deployed in natural watershed sites. The method used in this study was effective for monitoring E. coli O157:H7 in the farm environment, during postharvest processing, and in foodborne outbreak investigations.

Real-time PCR and enzyme-linked fluorescent assay methods for detecting Shiga-toxin-producingEscherichia coliin mincemeat samples

2007 ◽  
Vol 53 (3) ◽  
pp. 337-342 ◽  
Author(s):  
A. Stefan ◽  
S. Scaramagli ◽  
R. Bergami ◽  
C. Mazzini ◽  
M. Barbanera ◽  
...  
Keyword(s):  
Real Time ◽  
Shiga Toxin ◽  
Real Time Pcr ◽  
Central Italy ◽  
Health Concern ◽  
Bacterial Cells ◽  
Fluorescent Assay ◽  
Point Of Sale ◽  
E Coli ◽  
Assay Methods

This work aimed to compare real-time polymerase chain reaction (PCR) with the commercially available enzyme-linked fluorescent assay (ELFA) VIDAS ECOLI O157™ for detecting Escherichia coli O157 in mincemeat. In addition, a PCR-based survey on Shiga-toxin-producing E. coli (STEC) in mincemeat collected in Italy is presented. Real-time PCR assays targeting the stx genes and a specific STEC O157 sequence (SILO157, a small inserted locus of STEC O157) were tested for their sensitivity on spiked mincemeat samples. After overnight enrichment, the presence of STEC cells could be clearly determined in the 25 g samples containing 10 bacterial cells, while the addition of five bacteria provided equivocal PCR results with Ct values very close to or above the threshold of 40. The PCR tests proved to be more sensitive than the ELFA-VIDAS ECOLI O157™, whose detection level started from 50 bacterial cells/25 g of mincemeat. The occurrence of STEC in 106 mincemeat (bovine, veal) samples collected from September to November 2004 at five different points of sale in Italy (one point of sale in Arezzo, Tuscany, central Italy, two in Mantova, Lombardy, Northern Italy, and two in Bologna, Emilia-Romagna, upper-central Italy) was less than 1%. Contamination by the main STEC O-serogroups representing a major public health concern, including O26, O91, O111, O145, and O157, was not detected. This survey indicates that STEC present in these samples are probably not associated with pathogenesis in humans.

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