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Selective quantification of viable Escherichia coli bacteria in biosolids by quantitative PCR with propidium monoazide modification.

ABSTRACT: 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·liter(-1).

SUBMITTER: Taskin B

PROVIDER: S-EPMC3127682 | biostudies-literature | 2011 Jul

REPOSITORIES: biostudies-literature

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Selective quantification of viable Escherichia coli bacteria in biosolids by quantitative PCR with propidium monoazide modification.

Taskin Bilgin B Gozen Ayse Gul AG Duran Metin M

Applied and environmental microbiology 20110520 13

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 ...[more]

PMID: 21602375

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Project description:IntroductionToxigenic Vibrio cholerae serogroup O1 and O139 are the pathogens responsible for the global cholera epidemic. V. cholerae can settle in the water and spread via the fecal-oral route. Rapid and accurate monitoring of live V. cholerae in environmental water has become an important strategy to prevent and control cholera transmission. Conventional plate counting is widely used to detect viable bacteria but requires time and effort.MethodsThis study aims to develop a new assay that combines triplex droplet digital PCR (ddPCR) with propidium monoazide (PMA) treatment for quantitatively detecting live V. cholerae O1/O139 and cholera enterotoxin. Specific primers and probes were designed according to the conserved regions of gene rfb O1, rfb O139, and ctxA. The amplification procedures and PMA treatment conditions were optimized. The specificity, sensitivity, and ability of PMA-ddPCR to detect viable bacteria-derived DNA were evaluated in simulated seawater samples.Results and discussionThe results revealed that the optimal primer concentrations of rfb O1, rfb O139, and ctxA were 1 μM, while the concentrations of the three probes were 0.25, 0.25, and 0.4 μM, respectively. The best annealing temperature was 58°C to obtain the most accurate results. The optimal strategy for distinguishing dead and live bacteria from PMA treatment was incubation at the concentration of 20 μM for 15 min, followed by exposure to a 650-W halogen lamp for 20 min. In pure culture solutions, the limit of detection (LODs) of V. cholerae O1 and O139, and ctxA were 127.91, 120.23 CFU/mL, and 1.5 copies/reaction in PMA-triplex ddPCR, respectively, while the LODs of the three targets were 150.66, 147.57 CFU/mL, and 2 copies/reaction in seawater samples. The PMA-ddPCR sensitivity was about 10 times higher than that of PMA-qPCR. When detecting spiked seawater samples with live bacterial concentrations of 1.53 × 102 and 1.53 × 105 CFU/mL, the assay presented a higher sensitivity (100%, 16/16) than qPCR (50.00%, 8/16) and a perfect specificity (100%, 9/9). These results indicate that the developed PMA-triplex ddPCR is superior to the qPCR regarding sensitivity and specificity and can be used to rapidly detect viable toxigenic V. cholerae O1 and O139 in suspicious seawater samples.

Dataset Information

Selective quantification of viable Escherichia coli bacteria in biosolids by quantitative PCR with propidium monoazide modification.

Publications

Selective quantification of viable Escherichia coli bacteria in biosolids by quantitative PCR with propidium monoazide modification.

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