scholarly journals Reverse Transcription-Multiplex PCR Assay for Simultaneous Detection of Escherichia coli O157:H7, Vibrio cholerae O1, and Salmonella Typhi

2004 ◽  
Vol 50 (11) ◽  
pp. 2037-2044 ◽  
Author(s):  
Nicole J Morin ◽  
Zhilong Gong ◽  
Xing-Fang Li
Keyword(s):  
Escherichia Coli ◽  
Multiplex Pcr ◽  
Vibrio Cholerae ◽  
Reverse Transcription ◽  
Simultaneous Detection ◽  
Salmonella Typhi ◽  
Escherichia Coli O157 ◽  
Single Tube ◽  
E Coli ◽  
Vibrio Cholerae O1

Abstract Background: Escherichia coli O157:H7, Vibrio cholerae O1, and Salmonella Typhi are pathogenic bacteria that can be found in contaminated water supplies throughout the world. No currently available assays can simultaneously detect and identify all three pathogens. Our aim was to develop a rapid and reliable technique for simultaneous detection of these pathogens. Methods: Four unique genes were chosen as the targets of detection. Forward and reverse primers were designed to specifically amplify different sizes of these target genes: a 239-bp region of the E. coli O157 lipopolysaccharide (LPS) gene (rfbE); a 179-bp region of the H7 flagellin gene (fliC); a 419-bp region of the V. cholerae O1 LPS gene (rfbE); and a 329-bp region of Salmonella Typhi LPS gene (tyv). To ensure the detection of only viable replicating bacteria, RNA was extracted for analysis. After reverse transcription, cDNAs were simultaneously amplified in a single tube by multiplex PCR. The multiplex PCR products were analyzed by gel electrophoresis. To characterize the assay we analyzed, in a blinded fashion, seven unknown RNA samples containing various combinations of total RNA from these bacteria as well as clinical isolates. Results: All seven unknown RNA samples were correctly identified. The assay was able to detect and identify as few as 30 cells of E. coli O157:H7 and Salmonella Typhi in clinical isolates, and the presence of other bacteria did not interfere with the analysis. Conclusion: An assay combining reverse transcription with single-tube multiplex PCR was successfully developed and validated for simultaneous detection of viable E. coli O157:H7, V. cholerae O1, and Salmonella Typhi.

scholarly journals An Electrochemical Strategy using Multifunctional Nanoconjugates for Efficient Simultaneous Detection of Escherichia coli O157: H7 and Vibrio cholerae O1

Theranostics ◽  
2017 ◽  
Vol 7 (4) ◽  
pp. 935-944 ◽  
Author(s):  
Yan Li ◽  
Ya Xiong ◽  
Lichao Fang ◽  
Lili Jiang ◽  
Hui Huang ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Vibrio Cholerae ◽  
Simultaneous Detection ◽  
Escherichia Coli O157 ◽  
Vibrio Cholerae O1

Multiplex PCR for Simultaneous Detection of Salmonella spp., Listeria monocytogenes, and Escherichia coli O157:H7 in Meat Samples

2005 ◽  
Vol 68 (3) ◽  
pp. 551-556 ◽  
Author(s):  
SUSUMU KAWASAKI ◽  
NAOKO HORIKOSHI ◽  
YUKIO OKADA ◽  
KAZUKO TAKESHITA ◽  
TAKASHI SAMESHIMA ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Listeria Monocytogenes ◽  
Multiplex Pcr ◽  
Simultaneous Detection ◽  
Detection Sensitivity ◽  
Rapid Screening ◽  
Escherichia Coli O157 ◽  
Salmonella Spp ◽  
E Coli ◽  
Meat Samples

A multiplex PCR method was developed for simultaneous detection of Salmonella spp., Listeria monocytogenes, and Escherichia coli O157:H7 in meat samples. DNA detection sensitivity for this method was 103 CFU/ml for each pathogen. When this protocol was used for the detection of each of the above pathogenic bacteria in spiked pork samples, 1 cell per 25 g of inoculated sample could be detected within 30 h. In the samples of naturally contaminated meat, Salmonella spp., L. monocytogenes, and E. coli O157:H7 were detected over the same time period. Excellent agreement was obtained for the results of multiplex PCR and the conventional culture method, which suggests that the multiplex PCR is a reliable and useful method for rapid screening of meat products for Salmonella spp., L. monocytogenes, and E. coli O157:H7 contamination.

scholarly journals Evaluation of Multiplex PCR System for Simultaneous Detection of Escherichia coli O157:H7, Listeria monocytogenes and Salmonella enteritidis in Shrimp Samples

1970 ◽  
Vol 24 (1) ◽  
pp. 42-46 ◽  
Author(s):  
Mahmuda Yasmin ◽  
Susumu Kawasaki ◽  
Shinichi Kawamoto
Keyword(s):  
Escherichia Coli ◽  
Listeria Monocytogenes ◽  
Multiplex Pcr ◽  
Salmonella Enteritidis ◽  
Simultaneous Detection ◽  
Dna Amplification ◽  
Rapid Screening ◽  
Escherichia Coli O157 ◽  
E Coli ◽  
Pcr Method

A multiplex polymerase chain reaction (PCR) method was evaluated for simultaneous detection of Escherichia coli O157:H7, Listeria monocytogenes and Salmonella enteritidis in shrimp samples. The sensitivity of DNA amplification by PCR in this method was found to be 103 cfu/ml for each pathogen. When this protocol was adopted for the detection of each of the above mentioned pathogen in spiked shrimp extract culture, similar sensitivity was observed. However, this method detected 1 bacterial cell for E. coli O157:H7 and S. enteritidis and 100 for L. monocytogenes per 25 g spiked shrimp samples after overnight enrichment. In the commercially imported shrimp samples, none was found to contain any of the three pathogens by multiplex PCR or by conventional method, which suggests that the multiplex PCR is a reliable and useful for rapid screening of shrimp samples for E. coli O157:H7, L. monocytogenes and S. enteritidis. This will save time and increase our ability to assure food safety. Keywords: Multiples PCR, Shrimp extract, Spiked sample, Escherichia coli O157:H7, Listeria monocytogenes, Salmonella enteritidisDOI: http://dx.doi.org/10.3329/bjm.v24i1.1236 Bangladesh J Microbiol, Volume 24, Number 1, June 2007, pp 42-46

A gold nanoparticles-assisted multiplex PCR assay for simultaneous detection of Salmonella typhimurium, Listeria monocytogenes and Escherichia coli O157:H7

2020 ◽  
Vol 12 (2) ◽  
pp. 212-217 ◽  
Author(s):  
Juan Du ◽  
Shujing Wu ◽  
Liyuan Niu ◽  
Junguang Li ◽  
Dianbo Zhao ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Gold Nanoparticles ◽  
Listeria Monocytogenes ◽  
Salmonella Typhimurium ◽  
Multiplex Pcr ◽  
Simultaneous Detection ◽  
Escherichia Coli O157 ◽  
Pcr Assay ◽  
Multiplex Pcr Assay ◽  
Pcr Product

Unfunctionalized flower-shaped AuNPs is used as colorimetric sensor for PCR product detection by naked eyes.

scholarly journals Iha: a Novel Escherichia coli O157:H7 Adherence-Conferring Molecule Encoded on a Recently Acquired Chromosomal Island of Conserved Structure

2000 ◽  
Vol 68 (3) ◽  
pp. 1400-1407 ◽  
Author(s):  
Phillip I. Tarr ◽  
Sima S. Bilge ◽  
James C. Vary ◽  
Srdjan Jelacic ◽  
Rebecca L. Habeeb ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Epithelial Cells ◽  
Vibrio Cholerae ◽  
Shiga Toxin ◽  
Escherichia Coli O157 ◽  
Chromosomal Gene ◽  
Gene Encoding ◽  
Tellurite Resistance ◽  
E Coli ◽  
Dna Library

ABSTRACT The mechanisms used by Shiga toxin (Stx)-producingEscherichia coli to adhere to epithelial cells are incompletely understood. Two cosmids from an E. coliO157:H7 DNA library contain an adherence-conferring chromosomal gene encoding a protein similar to iron-regulated gene A (IrgA) ofVibrio cholerae (M. B. Goldberg, S. A. Boyko, J. R. Butterton, J. A. Stoebner, S. M. Payne, and S. B. Calderwood, Mol. Microbiol. 6:2407–2418, 1992). We have termed the product of this gene the IrgA homologue adhesin (Iha), which is encoded by iha. Iha is 67 kDa in E. coliO157:H7 and 78 kDa in laboratory E. coli and is structurally unlike other known adhesins. DNA adjacent toiha contains tellurite resistance loci and is conserved in structure in distantly related pathogenic E. coli, but it is absent from nontoxigenic E. coli O55:H7, sorbitol-fermenting Stx-producing E. coli O157:H−, and laboratory E. coli. We have termed this region the tellurite resistance- and adherence-conferring island. We conclude that Iha is a novel bacterial adherence-conferring protein and is contained within an E. coli chromosomal island of conserved structure. Pathogenic E. coli O157:H7 has only recently acquired this island.

Primers with 5′ Flaps Improve the Efficiency and Sensitivity of Multiplex PCR Assays for the Detection of Salmonella and Escherichia coli O157:H7

2013 ◽  
Vol 76 (4) ◽  
pp. 668-673 ◽  
Author(s):  
CHRIS TIMMONS ◽  
SHEFALI DOBHAL ◽  
JACQUELINE FLETCHER ◽  
LI MARIA MA
Keyword(s):  
Escherichia Coli ◽  
Multiplex Pcr ◽  
Fold Increase ◽  
Detection Sensitivity ◽  
Escherichia Coli O157 ◽  
Bacterial Cells ◽  
Robust Detection ◽  
E Coli ◽  
Pcr Multiplex ◽  
Pcr Assays

Foodborne illnesses caused by Salmonella enterica and Escherichia coli O157:H7 are worldwide health concerns. Rapid, sensitive, and robust detection of these pathogens in foods and in clinical and environmental samples is essential for routine food quality testing, effective surveillance, and outbreak investigations. The aim of this study was to evaluate the effect on PCR sensitivity of adding a short, AT-rich overhanging nucleotide sequence (flap) to the 5′ end of PCR primers specific for the detection of Salmonella and E. coli O157:H7. Primers targeting the invA gene of Salmonella and the rfbE gene of E. coli O157:H7 were synthesized with or without a 12-bp, AT-rich 5′ flap (5′-AATAAATCATAA-3′). Singleplex PCR, multiplex PCR, and real-time PCR sensitivity assays were conducted using purified bacterial genomic DNA and crude cell lysates of bacterial cells. The effect of background flora on detection was evaluated by spiking tomato and jalapeno pepper surface washes with E. coli O157:H7 and Salmonella Saintpaul. When targeting individual pathogens, end-point PCR assays using flap-amended primers were more efficient than nonamended primers, with 20.4 and 23.5% increases in amplicon yield for Salmonella and E. coli O157:H7, respectively. In multiplex PCR assays, a 10- to 100-fold increase in detection sensitivity was observed when the primer flap sequence was incorporated. This improvement in both singleplex and multiplex PCR efficiency and sensitivity can lead to improved Salmonella and E. coli O157:H7 detection.

A Multiplex Polymerase Chain Reaction Assay for Rapid Detection and Identification of Escherichia coli O157:H7 in Foods and Bovine Feces†

2000 ◽  
Vol 63 (8) ◽  
pp. 1032-1037 ◽  
Author(s):  
PINA M. FRATAMICO ◽  
LORI K. BAGI ◽  
TIZIANA PEPE
Keyword(s):  
Escherichia Coli ◽  
Polymerase Chain Reaction ◽  
Multiplex Pcr ◽  
Multiplex Polymerase Chain Reaction ◽  
Ground Beef ◽  
Escherichia Coli O157 ◽  
Chain Reaction ◽  
E Coli ◽  
Polymerase Chain ◽  
Bovine Feces

A multiplex polymerase chain reaction (PCR) assay was designed to simplify detection of Escherichia coli O157:H7 and to identify the H serogroup and the type of Shiga toxin produced by this bacterium. Primers for a plasmid-encoded hemolysin gene (hly933), and chromosomal flagella (fliCh7; flagellar structural gene of H7 serogroup), Shiga toxins (stx1, stx2), and attaching and effacing (eaeA) genes were used in a multiplex PCR for coamplification of the corresponding DNA sequences from enterohemorrhagic E. coli (EHEC) O157:H7. Enrichment cultures of ground beef, blue cheese, mussels, alfalfa sprouts, and bovine feces, artificially inoculated with various levels of E. coli O157:H7 strain 933, were subjected to a simple DNA extraction step prior to the PCR, and the resulting amplification products were analyzed by agarose gel electrophoresis. Sensitivity of the assay was ≤1 CFU/g of food or bovine feces (initial inoculum level), and results could be obtained within 24 h. Similar detection levels were obtained with ground beef samples that underwent enrichment culturing immediately after inoculation and samples that were frozen or refrigerated prior to enrichment. The multiplex PCR facilitates detection of E. coli O157:H7 and can reduce the time required for confirmation of isolates by up to 3 to 4 days.

scholarly journals Assessment of a multiplex detection method for Salmonella enterica, Escherichia coli O157:H7, and Listeria monocytogenes in cow milk

2019 ◽  
Vol 24 (1) ◽  
pp. 277-294
Author(s):  
Rocio Esperanza Patiño-Burbano ◽  
Ana Karina Carrascal ◽  
Jorge Luis Parra-Arango ◽  
José Luis Rodríguez-Bautista
Keyword(s):  
Escherichia Coli ◽  
Listeria Monocytogenes ◽  
Detection Rate ◽  
Pathogenic Bacteria ◽  
Simultaneous Detection ◽  
Cow Milk ◽  
Escherichia Coli O157 ◽  
Salmonella Spp ◽  
E Coli ◽  
Microbiological Methods

Raw cow milk is considered one of the most important vehicles for pathogenic bacteria like Salmonella spp., Escherichia coli O157:H7, and Listeria monocytogenes. These three bacteria are responsible for foodborne diseases. Routine microbiological methods to detect these microorganisms in cow milk can be complicated and time consuming. The aim of this work was to evaluate a method to simultaneously detect Salmonella spp., Escherichia coli O157:H7, and Listeria monocytogenes in experimentally contaminated cow milk. The assessed method combined a standard microbiological culture step, using a pre-enrichment medium that favors the growth of the three focal microorganisms: SEL broth, followed by a single PCR assay. A total of 43 interference bacterial strains were used to evaluate the method’s specificity. The detection rate for the microbiological method with standard culture media was 10 UFC/mL, and that of the PCR detection, following pre-enrichment in SEL broth, was 10 UFC/mL for S. enterica and L. monocytogenes and between 1 and 5 UFC/mL for E. coli O157:H7. The PCR method showed specificity for the reference strains. Simultaneous detection by multiple PCR using SEL broth was successful for the detection of S. enterica, E. coli O157:H7, and L. monocytogenes in samples of experimentally contaminated cow milk, featuring both a high detection rate and a high specificity. This approach promises to be a feasible routine procedure when testing milk samples in industry and public health control setups.

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