Comprehensive and Rapid Real-Time PCR Analysis of 21 Foodborne Outbreaks

A set of four duplex SYBR Green I PCR (SG-PCR) assay combined with DNA extraction using QIAamp DNA Stool Mini kit was evaluated for the detection of foodborne bacteria from 21 foodborne outbreaks. The causative pathogens were detected in almost all cases in 2 hours or less. The first run was for the detection of 8 main foodborne pathogens in 5 stool specimens within 2 hours and the second run was for the detection of other unusual suspect pathogens within a further 45 minutes. After 2 to 4 days, the causative agents were isolated and identified. The results proved that for comprehensive and rapid molecular diagnosis in foodborne outbreaks, Duplex SG-PCR assay is not only very useful, but is also economically viable for one-step differentiation of causative pathogens in fecal specimens obtained from symptomatic patients. This then allows for effective diagnosis and management of foodborne outbreaks.

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Simultaneous Screening of 24 Target Genes of Foodborne Pathogens in 35 Foodborne Outbreaks Using Multiplex Real-Time SYBR Green PCR Analysis

International Journal of Microbiology ◽

10.1155/2010/864817 ◽

2010 ◽

Vol 2010 ◽

pp. 1-18 ◽

Cited By ~ 24

Author(s):

Hiroshi Fukushima ◽

Jun Kawase ◽

Yoshiki Etoh ◽

Kumiko Sugama ◽

Shunshuke Yashiro ◽

...

Keyword(s):

Real Time ◽

Foodborne Pathogens ◽

Target Genes ◽

Simultaneous Detection ◽

Sybr Green ◽

Pcr Analysis ◽

Foodborne Outbreak ◽

Pcr Assay ◽

Foodborne Outbreaks ◽

Stool Specimens

A set of 8 multiplex real-time SYBR Green PCR (SG-PCR) assays including 3 target primers and an internal amplification control (IAC) primer was simultaneously evaluated in 3 h or less with regard to detection of 24 target genes of 23 foodborne pathogens in 7 stool specimens of foodborne outbreak using a 96-well reaction plate. This assay, combined with DNA extraction (QIAamp DNA Stool Mini kit), offered detection of greater than103-104foodborne pathogens per g in stool specimens. The products formed were identified using melting point temperature (Tm) curve analysis. This assay was evaluated for the detection of foodborne pathogens in 33 out of 35 cases of foodborne outbreak, using 4 different PCR instruments in 5 different laboratories. No interference from the multiplex real-time SG-PCR assay, including IAC, was observed in stool specimens in any analysis. We found multiplex real-time SG-PCR assay for simultaneous detection of 24 target genes of foodborne pathogens to be comprehensive, rapid, inexpensive, accurate, of high selectivity, and good for screening probability.

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Improvement in the detection rate of diarrhoeagenic bacteria in human stool specimens by a rapid real-time PCR assay

Journal of Medical Microbiology ◽

10.1099/jmm.0.45607-0 ◽

2004 ◽

Vol 53 (7) ◽

pp. 617-622 ◽

Cited By ~ 46

Author(s):

Yoshio Iijima ◽

Nahoko T. Asako ◽

Masanori Aihara ◽

Kozaburo Hayashi

Keyword(s):

Real Time ◽

Real Time Pcr ◽

Bacterial Infections ◽

Food Poisoning ◽

Culture Method ◽

Pcr Analysis ◽

Laboratory System ◽

Pcr Assay ◽

Conventional Culture ◽

Stool Specimens

A rapid laboratory system has been developed and evaluated that can simultaneously identify major diarrhoeagenic bacteria, including Salmonella enterica, Vibrio parahaemolyticus, Campylobacter jejuni and Shiga toxin-producing Escherichia coli, in stool specimens by real-time PCR. Specific identification was achieved by using selective TaqMan probes, detecting two targets in each pathogen. A positive result was scored only when both targets of a pathogen were amplified and the difference between threshold cycles for detection was less than five. Diagnosis of enteric bacterial infections using this highly sensitive method, including DNA extraction and real-time PCR, requires only 3 h. Forty stool specimens related to suspected food poisoning outbreaks were analysed: 16 (40 %) of these samples were found to be positive for diarrhoeagenic bacteria using a conventional culture method; 28 (70 %) were positive using the real-time PCR assay. Of the 12 PCR-positive but culture-negative cases, 11 patients had consumed pathogen-contaminated or high-risk food. Analysis of faecal samples from 105 outpatients who complained of diarrhoea and/or abdominal pain identified 19 (18 %) patients as being positive for diarrhoeagenic bacteria using the culture method. An additional six (6 %) patients were found to be positive by PCR analysis.

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Faculty Opinions recommendation of One-step, multiplex, real-time PCR assay with molecular beacon probes for simultaneous detection, differentiation, and quantification of human T-cell leukemia virus types 1, 2, and 3.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.713998082.789502847 ◽

2012 ◽

Author(s):

Sanjay Tyagi

Keyword(s):

T Cell ◽

Real Time Pcr ◽

Molecular Beacon ◽

Leukemia Virus ◽

Simultaneous Detection ◽

Cell Leukemia ◽

Pcr Assay ◽

Human T Cell ◽

T Cell Leukemia Virus ◽

One Step

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A One-Step, Real-Time PCR Assay for Rapid Detection of Rhinovirus

Journal of Molecular Diagnostics ◽

10.2353/jmoldx.2010.090071 ◽

2010 ◽

Vol 12 (1) ◽

pp. 102-108 ◽

Cited By ~ 16

Author(s):

Duc H. Do ◽

Stella Laus ◽

Amy Leber ◽

Mario J. Marcon ◽

Jeanne A. Jordan ◽

...

Keyword(s):

Real Time ◽

Real Time Pcr ◽

Rapid Detection ◽

Pcr Assay ◽

One Step

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One-step real-time PCR assay for detection and quantification of RNA HCV to monitor patients under treatment in Brazil

The Brazilian Journal of Infectious Diseases ◽

10.1016/j.bjid.2018.08.003 ◽

2018 ◽

Vol 22 (5) ◽

pp. 418-423

Author(s):

Elisabete Andrade ◽

Daniele Rocha ◽

Marcela Fontana-Maurell ◽

Elaine Costa ◽

Marisa Ribeiro ◽

...

Keyword(s):

Real Time ◽

Real Time Pcr ◽

Pcr Assay ◽

One Step ◽

Detection And Quantification

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Detection of Mycoplasma hyopneumoniae by Air Sampling with a Nested PCR Assay

Applied and Environmental Microbiology ◽

10.1128/aem.64.2.543-548.1998 ◽

1998 ◽

Vol 64 (2) ◽

pp. 543-548 ◽

Cited By ~ 78

Author(s):

Katharina D. C. Stärk ◽

Jacques Nicolet ◽

Joachim Frey

Keyword(s):

Nested Pcr ◽

Gene Segment ◽

Mycoplasma Hyopneumoniae ◽

Field Conditions ◽

Pcr Analysis ◽

Direct Extraction ◽

Pcr Assay ◽

Sampling System ◽

Respiratory Problems ◽

One Step

ABSTRACT This article describes the first successful detection of airborneMycoplasma hyopneumoniae under experimental and field conditions with a new nested PCR assay. Air was sampled with polyethersulfone membranes (pore size, 0.2 μm) mounted in filter holders. Filters were processed by dissolution and direct extraction of DNA for PCR analysis. For the PCR, two nested pairs of oligonucleotide primers were designed by using an M. hyopneumoniae-specific DNA sequence of a repeated gene segment. A nested PCR assay was developed and used to analyze samples collected in eight pig houses where respiratory problems had been common. Air was also sampled from a mycoplasma-free herd. The nested PCR was highly specific and 104 times as sensitive as a one-step PCR. Under field conditions, the sampling system was able to detect airborne M. hyopneumoniae on 80% of farms where acute respiratory disease was present. No airborne M. hyopneumoniae was detected on infected farms without acute cases. The chance of successful detection was increased if air was sampled at several locations within a room and at a lower air humidity.

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Developing a multiplex real-time PCR with a new pre-enrichment to simultaneously detect four foodborne bacteria in milk

Future Microbiology ◽

10.2217/fmb-2019-0044 ◽

2019 ◽

Vol 14 (10) ◽

pp. 885-898 ◽

Cited By ~ 2

Author(s):

Moezi Parichehr ◽

Kargar Mohammad ◽

Doosti Abbas ◽

Khoshneviszadeh Mehdi

Keyword(s):

Real Time ◽

Foodborne Pathogens ◽

Real Time Pcr ◽

Simultaneous Detection ◽

Pcr Assay ◽

Reliable Technique ◽

E Coli ◽

Foodborne Bacteria ◽

Multiple Pathogens ◽

Enrichment Step

Aim: The aim of this study is to formulate a new single nonselective pre-enrichment medium (ELSS) that can support the concurrent growth of four major foodborne pathogens containing E. coli O157: H7, L. monocytogenes, S. aureus and S. enterica serovar Entertidis to develop a multiplex TaqMan Real-time PCR (mRT-PCR). Methods: The mRT-PCR with a new pre-enrichment was carried out for simultaneous detection and quantification of these foodborne bacteria. Results: By using mRT-PCR after 16 h pre-enrichment in ELSS, the detection limit of each pathogen was 1 CFU/25 ml contaminated milk, as well as inclusivity and exclusivity reached 100%. Conclusion: The mRT-PCR assay with pre-enrichment step is a fast and reliable technique for detecting single or multiple pathogens in food products.

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