Method To Detect Only Live Bacteria during PCR Amplification

Soejima, Takashi; Iida, Keiichiro; Qin, Tian; Taniai, Hiroaki; Seki, Masanori; Yoshida, Shigeaki

doi:10.1128/jcm.02171-07

Cited by 86 publications

(82 citation statements)

References 37 publications

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“…It is possible that the amplicon's being too short prevented complete chelation by PMA and the non-cross-linked DNA was amplified. Soejima et al (34) indicated that EMA could not completely inhibit DNA amplification when the target DNA was short (113 bp), while PCR amplification of long DNA (894 bp in the 23S rRNA gene) was suppressed by EMA more than that of short DNA (113 bp in the hly gene). Also, Luo et al (17) recently showed that PMA treatment cannot efficiently suppress dead cells from PCR amplification when the targeted gene is as short as 190 bp.…”

Section: Discussionmentioning

confidence: 99%

Selective Quantification of Viable Escherichia coli Bacteria in Biosolids by Quantitative PCR with Propidium Monoazide Modification

Taşkın

Gözen

Duran

2011

Appl Environ Microbiol

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Quantitative differentiation of live cells in biosolids samples, without the use of culturing-based approaches, is highly critical from a public health risk perspective, as recent studies have shown significant regrowth and reactivation of indicator organisms. Persistence of DNA in the environment after cell death in the range of days to weeks limits the application of DNA-based approaches as a measure of live cell density. Using selective nucleic acid intercalating dyes like ethidium monoazide (EMA) and propidium monoazide (PMA) is one of the alternative approaches to detecting and quantifying viable cells by quantitative PCR. These compounds have the ability to penetrate only into dead cells with compromised membrane integrity and intercalate with DNA via their photoinducible azide groups and in turn inhibit DNA amplification during PCRs. PMA has been successfully used in different studies and microorganisms, but it has not been evaluated sufficiently for complex environmental samples such as biosolids. In this study, experiments were performed with Escherichia coli ATCC 25922 as the model organism and the uidA gene as the target sequence using real-time PCR via the absolute quantification method. Experiments with the known quantities of live and dead cell mixtures showed that PMA treatment inhibits PCR amplification from dead cells with over 99% efficiency. The results also indicated that PMA-modified quantitative PCR could be successfully applied to biosolids when the total suspended solids (TSS) concentration is at or below 2,000 mg ⅐ liter ؊1 .

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Section: Discussionmentioning

confidence: 99%

Selective Quantification of Viable Escherichia coli Bacteria in Biosolids by Quantitative PCR with Propidium Monoazide Modification

Taşkın

Gözen

Duran

2011

Appl Environ Microbiol

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“…Several published articles outline the various approaches that have been used to detect viable cells in combination with nucleic acid amplification methods which include the following: -PMA solution ranging from 6.25 mM [22] to 50 mM [23] to 400 mM [15], where the authors obtained signal reductions ranging from 3.85 to 5 log units. -Incubation periods in the dark ranging from 5 min [19,15,24] to 20 min [25] and light exposure from 2 min [23] to 20 min [26]. -Photoactivation carried out using high-power halogen lamps ranging from 500 to 750 W [23,27] and samples placed horizontally on ice a distance of 20e30 cm from the light source [19,24,28].…”

Section: Introductionmentioning

confidence: 99%

Legionella in water samples: How can you interpret the results obtained by quantitative PCR?

Ditommaso

Ricciardi

Giacomuzzi

et al. 2015

Molecular and Cellular Probes

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“…Nocker et al (41,42) suggested that the signal reduction was due to a selective loss of genomic DNA from dead cells (rendered insoluble after cross-linkage) during the DNA extraction procedure rather than to PCR inhibition. However, Soejima et al (59,60) recently reported that treatment with EMA followed by visible light irradiation directly cleaves the chromosomal DNA of dead bacteria.…”

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confidence: 99%

Viability PCR, a Culture-Independent Method for Rapid and Selective Quantification of Viable Legionella pneumophila Cells in Environmental Water Samples

Delgado‐Viscogliosi

Solignac

Delattre

2009

Appl Environ Microbiol

134

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Method To Detect Only Live Bacteria during PCR Amplification

Cited by 86 publications

References 37 publications

Selective Quantification of Viable Escherichia coli Bacteria in Biosolids by Quantitative PCR with Propidium Monoazide Modification

Selective Quantification of Viable Escherichia coli Bacteria in Biosolids by Quantitative PCR with Propidium Monoazide Modification

Legionella in water samples: How can you interpret the results obtained by quantitative PCR?

Viability PCR, a Culture-Independent Method for Rapid and Selective Quantification of Viable Legionella pneumophila Cells in Environmental Water Samples

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