Template DNA Ratio can Affect Detection by Genus‐Specific PCR–Denaturing Gradient Gel Electrophoresis of Bacteria Present at Low Abundance in Mixed Populations

Zhang, Li; Danon, Stephen J.; Grehan, Martin; Lee, Adrian V.; Mitchell, Hazel M.

doi:10.1111/j.1523-5378.2005.00294.x

Cited by 7 publications

(4 citation statements)

References 8 publications

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“…Thus, when the concentration of the DNA template of a particular bacterial species is signiWcantly higher than that of another species, the species with a lower concentration of the DNA template may not be detected by PCR-DGGE even if this "low" concentration is higher than the detection limit of single PCR. Zhang et al have reported that the minimum ratio was between 1:20 and 1:50 when the lower DNA was not detected in the PCR-DGGE using two bacterial DNA as the template [14]. Bollmann et al also performed a similar experiment and have shown the ratio at which the lower DNA would not be detected to be between 1:29 and 1:99 [13].…”

Section: Discussionmentioning

confidence: 91%

“…PCR is known to have high detection sensitivity. However, it has been reported that the initial template DNA ratio in a sample obtained from a mixed population may aVect the detection of bacterial species that have low levels of abundance by genus-speciWc PCR-DGGE [13,14]. Thus, when the concentration of the DNA template of a particular bacterial species is signiWcantly higher than that of another species, the species with a lower concentration of the DNA template may not be detected by PCR-DGGE even if this "low" concentration is higher than the detection limit of single PCR.…”

Section: Discussionmentioning

confidence: 99%

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Change in the community structure of ammonia-oxidizing bacteria in activated sludge during selective incubation for MPN determination

Hirooka

Asano

Nakai

2009

J Ind Microbiol Biotechnol

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We investigated the changes in the community structure of ammonia-oxidizing bacteria (AOB) in activated sludge during incubation of the sludge in a medium selective for AOB. The number of AOB present in the activated sludge sample was enumerated by the most-probable-number (MPN) method. Both the activated sludge sample and the incubated samples for MPN determination were analyzed by polymerase chain reaction and denaturing gradient gel electrophoresis (PCR-DGGE). Universal PCR-DGGE indicated that even after 40-d incubation in a medium selected for AOB, the MPN samples were predominantly composed of heterotrophic bacteria and not AOB. Denitrification by heterotrophic bacteria might lead to the underestimation of the MPN count of AOB. Not dominated in whole bacteria, one species of AOB was detected in both original activated sludge and samples after MPN incubation by PCR-DGGE targeting AOB. Furthermore, two new species of AOB were detected only after incubation. Therefore, the community structure of AOB in the MPN samples partially resembled that in the original activated sludge.

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Section: Discussionmentioning

confidence: 91%

Section: Discussionmentioning

confidence: 99%

Change in the community structure of ammonia-oxidizing bacteria in activated sludge during selective incubation for MPN determination

Hirooka

Asano

Nakai

2009

J Ind Microbiol Biotechnol

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“…For bacteria, changes were real but not clearly correlated with grape development. This may result from a variation of species ratio and the detection of highly abundant species in a mixed population (Zhang et al 2005). Climatic variations (temperature, humidity, UV radiation), agrochemical applications, and viticultural practices such as leaf thinning, are several factors that are liable to influence the berry micro-environment and consequently the microbial ecosystem on the surface.…”

Section: Discussionmentioning

confidence: 99%

Understanding the microbial ecosystem on the grape berry surface through numeration and identification of yeast and bacteria

Renouf¹,

Claisse²,

Lonvaud-Funel³

2005

Aust J Grape Wine Res

187

147

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Microbial species present on the surface of grape berries at harvest play an important role in winemaking, thus counting and identifying them is of great importance. The use of conventional microbial techniques and molecular methods allowed a quantitative and qualitative inventory of the different microbial species present on the grape berries. These experiments were carried out in several areas of the Bordeaux region on the red grape varieties Merlot, Cabernet Sauvignon and Cabernet Franc. Populations and species clearly varied according to berry development stage. The most widespread yeast species at berry set, Aureobasidium pullulans was never detected at harvest. Fermentative yeasts were detected at harvest and not in the first stage of grape growth. Oenoccocus oeni was detected on immature as well as on mature berries. Gluconobacter oxydans was detected mainly at harvest. Detection of Pediococcus parvulus, was dependent on the vineyard. Veraison appeared to be a key stage for yeast colonisation and the increase in population involved a change in the proportion of each species. The number of A. pullulans fell significantly at veraison as it was superseded by fermentative yeasts. Microbial populations peaked at harvest when the berry surface available for adhesion was largest and no agrochemical treatments had been applied for some weeks. Soil, grape variety and grapegrowing practices may also influence this microbial ecosystem. Based on these and published data, we formulated hypotheses to describe this microbial ecosystem, thus enabling us to develop the concept of a microbial biofilm.

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“…In general, the potential to detect a specific taxon can be improved by using group-specific primers that can narrow the size of the target population. Even when using a genus-specific primer, however, the template DNA ratio may still affect the DGGE-based detection of certain species that are underrepresented in a mixed community sample [128]. Although poorly studied for SDE-based community fingerprinting of human microbiota, multiple displacement amplification (MDA) may provide another strategy to enhance the detection level especially in biopsy samples with lower bacterial counts.…”

Section: Limitations and Potential Pitfallsmentioning

confidence: 99%

Application of Sequence-Dependent Electrophoresis Fingerprinting in Exploring Biodiversity and Population Dynamics of Human Intestinal Microbiota: What Can Be Revealed?

Huys

Vanhoutte

Vandamme

2008

Interdisciplinary Perspectives on Infectious Diseases

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Sequence-dependent electrophoresis (SDE) fingerprinting techniques such as denaturing gradient gel electrophoresis (DGGE) have become commonplace in the field of molecular microbial ecology. The success of the SDE technology lays in the fact that it allows visualization of the predominant members of complex microbial ecosystems independent of their culturability and without prior knowledge on the complexity and diversity of the ecosystem. Mainly using the prokaryotic 16S rRNA gene as PCR amplification target, SDE-based community fingerprinting turned into one of the leading molecular tools to unravel the diversity and population dynamics of human intestinal microbiota. The first part of this review covers the methodological concept of SDE fingerprinting and the technical hurdles for analyzing intestinal samples. Subsequently, the current state-of-the-art of DGGE and related techniques to analyze human intestinal microbiota from healthy individuals and from patients with intestinal disorders is surveyed. In addition, the applicability of SDE analysis to monitor intestinal population changes upon nutritional or therapeutic interventions is critically evaluated.

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Template DNA Ratio can Affect Detection by Genus‐Specific PCR–Denaturing Gradient Gel Electrophoresis of Bacteria Present at Low Abundance in Mixed Populations

Cited by 7 publications

References 8 publications

Change in the community structure of ammonia-oxidizing bacteria in activated sludge during selective incubation for MPN determination

Change in the community structure of ammonia-oxidizing bacteria in activated sludge during selective incubation for MPN determination

Understanding the microbial ecosystem on the grape berry surface through numeration and identification of yeast and bacteria

Application of Sequence-Dependent Electrophoresis Fingerprinting in Exploring Biodiversity and Population Dynamics of Human Intestinal Microbiota: What Can Be Revealed?

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