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Detection of Live Escherichia coli O157:H7 Cells by PMA-qPCR
Published on: February 1, 2014
Evaluation of the Propidium Monoazide-quantitative Polymerase Chain Reaction Method for the Detection of Viable
Ericka T Pinheiro1, Viviane D Neves1, Caroline C Reis1
1Discipline of Endodontics, Department of Dentistry, School of Dentistry, University of São Paulo, São Paulo, Brazil.
Introduction:
One limitation of DNA-based molecular assays is their inability to distinguish between live and dead cells. A sample treatment with propidium monoazide (PMA) before DNA amplification has been proposed to overcome this problem. The aim of this in vitro study was to test different concentrations of PMA coupled with quantitative polymerase chain reaction (qPCR) for the detection of viable Enterococcus faecalis.
Methods:
Viable or heat-killed suspensions of E. faecalis (10⁶ colony-forming units/mL) were treated with PMA at 10, 50, and 100 μg/mL before DNA extraction. qPCR was performed using primers complementary for E. faecalis 16S ribosomal RNA sequence. PMA was also tested on bacteria suspensions containing different proportions of viable and dead cells. Bacterial suspensions without PMA treatment were used as positive controls.
Results:
The treatment of heat-killed suspensions with PMA at different concentrations significantly reduced the DNA amplification when compared with the group without treatment (P < .0001), indicating that DNA from dead cells was not used as templates. The greatest reduction in qPCR amplification of dead cell DNA was found when 100 μg/mL PMA was used (P < .005). In mixtures containing live/dead cells, PMA allowed selective detection of viable cells.
Conclusions:
PMA was effective in inhibiting qPCR amplification from the DNA of dead cells, enabling in vitro detection and quantification of viable cells of E. faecalis.
Insights
Propidium monoazide (PMA) effectively distinguishes live from dead Enterococcus faecalis cells in DNA assays. This method enables accurate quantification of viable bacteria by inhibiting DNA amplification from dead cells.
Area of Science:
- Microbiology
- Molecular Biology
Background:
- DNA-based molecular assays struggle to differentiate between live and dead cells.
- Propidium monoazide (PMA) treatment before DNA amplification is a proposed solution.
- This study investigates PMA efficacy for viable Enterococcus faecalis detection.
Purpose of the Study:
- To evaluate the effectiveness of different propidium monoazide (PMA) concentrations.
- To assess PMA's ability to inhibit DNA amplification from dead Enterococcus faecalis cells.
- To enable selective detection and quantification of viable Enterococcus faecalis using qPCR.
Main Methods:
- Bacterial suspensions (viable and heat-killed E. faecalis) were treated with PMA (10, 50, 100 μg/mL).
- DNA extraction followed by quantitative polymerase chain reaction (qPCR) targeting the 16S ribosomal RNA sequence.
- PMA efficacy was tested on mixed viable/dead cell suspensions and compared to untreated controls.
Main Results:
- PMA treatment significantly reduced DNA amplification from heat-killed E. faecalis (P < .0001).
- The highest PMA concentration (100 μg/mL) showed the greatest inhibition of dead cell DNA amplification (P < .005).
- PMA enabled selective detection of viable cells in mixtures of live and dead bacteria.
Conclusions:
- Propidium monoazide (PMA) effectively inhibits qPCR amplification from dead cell DNA.
- PMA facilitates accurate in vitro detection and quantification of viable Enterococcus faecalis.
- This approach overcomes a key limitation in DNA-based microbial detection.
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