Risk assessment of false-positive quantitative real-time PCR results in food, due to detection of DNA originating from dead cells

Wolffs, Petra; Norling, Börje; Rådström, Peter

doi:10.1016/j.mimet.2004.10.003

Cited by 101 publications

(57 citation statements)

References 27 publications

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“…Alternatively, Rothia may have been dormant in the sputum samples at the time of collection, its DNA in the sputum amplified by PCR from either dead cells or extracellular sources. The WinCF system may enrich for actively growing microbes, and future work is needed to investigate discrepancies between active microbes in a sputum sample and microbiome profiles by 16S rRNA gene amplification, which can be susceptible to sequencing 'dead' DNA (Wolffs et al, 2005;Young et al, 2007;Rogers et al, 2010).…”

Section: Sources Of the Fermentative Responsementioning

confidence: 99%

A Winogradsky-based culture system shows an association between microbial fermentation and cystic fibrosis exacerbation

et al. 2014

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There is a poor understanding of how the physiology of polymicrobial communities in cystic fibrosis (CF) lungs contributes to pulmonary exacerbations and lung function decline. In this study, a microbial culture system based on the principles of the Winogradsky column (WinCF system) was developed to study the physiology of CF microbes. The system used glass capillary tubes filled with artificial sputum medium to mimic a clogged airway bronchiole. Chemical indicators were added to observe microbial physiology within the tubes. Characterization of sputum samples from seven patients showed variation in pH, respiration, biofilm formation and gas production, indicating that the physiology of CF microbial communities varied among patients. Incubation of homogenized tissues from an explant CF lung mirrored responses of a Pseudomonas aeruginosa pure culture, supporting evidence that end-stage lungs are dominated by this pathogen. Longitudinal sputum samples taken through two exacerbation events in a single patient showed that a two-unit drop in pH and a 30% increase in gas production occurred in the tubes prior to exacerbation, which was reversed with antibiotic treatment. Microbial community profiles obtained through amplification and sequencing of the 16S rRNA gene showed that fermentative anaerobes became more abundant during exacerbation and were then reduced during treatment where P. aeruginosa became the dominant bacterium. Results from the WinCF experiments support the model where two functionally different CF microbial communities exist, the persistent Climax Community and the acute Attack Community. Fermentative anaerobes are hypothesized to be the core members of the Attack Community and production of acidic and gaseous products from fermentation may drive developing exacerbations. Treatment targeting the Attack Community may better resolve exacerbations and resulting lung damage.

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Section: Sources Of the Fermentative Responsementioning

confidence: 99%

A Winogradsky-based culture system shows an association between microbial fermentation and cystic fibrosis exacerbation

et al. 2014

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“…E. coli O157:H7 has the ability to readily adapt to and survive in a wide range of environmental conditions, including temperature changes, low pH, and desiccation (2). Furthermore, it has been observed that DNA from dead cells could be detected over a period of 28 days from cell death by three different detection methodologies (38). A number of conventional PCR and real-time PCR methodologies have been reported for the detection of E. coli O157:H7, but these PCR assays detect total DNA derived from both viable and dead cells and, thus, no determination can be made about the presence of only viable cells in the samples (15,35).…”

Section: Discussionmentioning

confidence: 99%

Real-Time PCR Methodology for Selective Detection of Viable Escherichia coli O157:H7 Cells by Targeting Z3276 as a Genetic Marker

Chen

2012

Appl Environ Microbiol

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The goal of this study was to develop a sensitive, specific, and accurate method for the selective detection of viable Escherichia coli O157:H7 cells in foods. A unique open reading frame (ORF), Z3276, was identified as a specific genetic marker for the detection of E. coli O157:H7. We developed a real-time PCR assay with primers and probe targeting ORF Z3276 and confirmed that this assay was sensitive and specific for E. coli O157:H7 strains (n ‫؍‬ 298). Using this assay, we can detect amounts of genomic DNA of E. coli O157:H7 as low as a few CFU equivalents. Moreover, we have developed a new propidium monoazide (PMA)-real-time PCR protocol that allows for the clear differentiation of viable from dead cells. In addition, the protocol was adapted to a 96-well plate format for easy and consistent handling of a large number of samples. Amplification of DNA from PMA-treated dead cells was almost completely inhibited, in contrast to the virtually unaffected amplification of DNA from PMA-treated viable cells. With beef spiked simultaneously with 8 ؋ 10 7 dead cells/g and 80 CFU viable cells/g, we were able to selectively detect viable E. coli O157:H7 cells with an 8-h enrichment. In conclusion, this PMA-real-time PCR assay offers a sensitive and specific means to selectively detect viable E. coli O157:H7 cells in spiked beef. It also has the potential for high-throughput selective detection of viable E. coli O157:H7 cells in other food matrices and, thus, will have an impact on the accurate microbiological and epidemiological monitoring of food safety and environmental sources. E scherichia coli O157:H7 is a major food-borne pathogen responsible for a number of outbreaks in animals, poultry, and humans worldwide (1,7,14). It is estimated that E. coli O157:H7 outbreaks infect over 73,500 people annually in the United States (24), and this pathogen has not only become an important food safety concern but also a serious medical and public health problem. In order to effectively handle future outbreaks in a timely manner, it is necessary to have ample availability of sensitive, specific, and reliable methodologies that can be used for the rapid and selective detection of E. coli O157:H7.To date, great efforts have been made to develop appropriate methodologies for the detection of E. coli O157:H7. The traditional culture methods use selective media or chromogenic agar to detect E. coli O157:H7 (23). However, major limitations of this method are that it takes days to obtain the culture while providing little accurate information about the nature of the strain or isolate itself (39). PCR has been widely used for the detection of E. coli O157:H7 from foods and environmental samples. More recently, real-time PCR is gaining popularity for its enhanced sensitivity and specificity and its speedy turnaround time. Numerous real-time PCR-based methods have been reported for rapid and sensitive detection of E. coli O157:H7 (4, 7, 9, 10). One of the major obstacles for the PCR-based methodologies for sensitive and specific detection...

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“…Another problem of direct PCR analysis from sample material is the possible detection of dead cells due to intact DNA after cell lysis (218). Enrichment procedures partly solve the problem by increasing the number of live cells in proportion to dead cells (98).…”

Section: Molecular Detection Of Clostridium Botulinummentioning

confidence: 99%

Laboratory Diagnostics of Botulism

2006

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SUMMARY Botulism is a potentially lethal paralytic disease caused by botulinum neurotoxin. Human pathogenic neurotoxins of types A, B, E, and F are produced by a diverse group of anaerobic spore-forming bacteria, including Clostridium botulinum groups I and II, Clostridium butyricum, and Clostridium baratii. The routine laboratory diagnostics of botulism is based on the detection of botulinum neurotoxin in the patient. Detection of toxin-producing clostridia in the patient and/or the vehicle confirms the diagnosis. The neurotoxin detection is based on the mouse lethality assay. Sensitive and rapid in vitro assays have been developed, but they have not yet been appropriately validated on clinical and food matrices. Culture methods for C. botulinum are poorly developed, and efficient isolation and identification tools are lacking. Molecular techniques targeted to the neurotoxin genes are ideal for the detection and identification of C. botulinum, but they do not detect biologically active neurotoxin and should not be used alone. Apart from rapid diagnosis, the laboratory diagnostics of botulism should aim at increasing our understanding of the epidemiology and prevention of the disease. Therefore, the toxin-producing organisms should be routinely isolated from the patient and the vehicle. The physiological group and genetic traits of the isolates should be determined.

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Risk assessment of false-positive quantitative real-time PCR results in food, due to detection of DNA originating from dead cells

Cited by 101 publications

References 27 publications

A Winogradsky-based culture system shows an association between microbial fermentation and cystic fibrosis exacerbation

A Winogradsky-based culture system shows an association between microbial fermentation and cystic fibrosis exacerbation

Real-Time PCR Methodology for Selective Detection of Viable Escherichia coli O157:H7 Cells by Targeting Z3276 as a Genetic Marker

Laboratory Diagnostics of Botulism

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