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Optimization of a propidium monoazide-qPCR method for Escherichia coli quantification in raw seafood
Marilia Miotto1, Clarissa Barretta1, Sylvia O Ossai2
1Department of Food Science and Technology, Federal University of Santa Catarina, 1346 Admar Gonzaga, 88034-001 Florianopolis, Santa Catarina, Brazil.
Abstract:
The present study compared different concentrations of propidium monoazide (PMA), time of exposure to light and different light intensities to determine the optimal conditions for the quantification of viable Escherichia coli in cell suspension and in food matrix. The influence of cell density and the effectiveness of PMA in viable but non-culturable (VBNC) E. coli cells were evaluated and also applied in food matrix. For that purpose, different concentrations of PMA (20 μM, 40 μM, 50 μM, 60 μM and 80 μM) under different times of exposure (5 min, 10 min, 15 min, 20 min and 30 min) to lights of different intensities (500 W and 650 W) were evaluated. After determining the optimal conditions, the PMA-qPCR methods were applied to different compositions of live and heat-killed E. coli suspensions (v:v; 0:1; 1:0; 1:1) in concentrations ranging from 3 Log to 7 Log CFU/mL. The same dilutions were prepared with E. coli in VBNC state and applied in food matrix. The results obtained from qPCR, PMA-qPCR and plate counts were compared. The results suggested that a PMA treatment of 50 μM PMA for 15 min under 650 W light intensity was optimal under our conditions. For E. coli cell suspensions, the amplification of heat-killed cells was inhibited greatly by PMA when concentrations were ≤ 5 Log CFU/mL. For the samples of oyster inoculated with heat-killed cells, E. coli was not detected by PMA-qPCR in concentrations ≤4 Log CFU/g. Regarding the results with VBNC state, we considered the PMA-qPCR method to be applicable for enumerating E. coli VBNC cells in oyster samples. Based on our findings, we further recommend the use of PMA-qPCR with the aim of reducing the amplification of dead cells for improving its performance, since false-positives could still occur depending on the level of E. coli in the sample. The application of the PMA-qPCR for quantification of bacteria, compared to the use of culture-dependent methods, is quite promising. However, further studies are recommended, especially using different food matrices.
Insights
This study optimized propidium monoazide quantitative PCR (PMA-qPCR) for viable Escherichia coli detection. Optimal conditions (50 μM PMA, 15 min, 650 W) effectively differentiated live from dead cells, showing promise for food safety applications.
Area of Science:
- Microbiology
- Molecular Biology
- Food Science
Background:
- Accurate quantification of viable bacteria like Escherichia coli is crucial for food safety.
- Traditional culture-dependent methods can be time-consuming and may not detect viable but non-culturable (VBNC) cells.
- Quantitative PCR (qPCR) can rapidly detect bacterial DNA, but often amplifies DNA from both live and dead cells, leading to overestimation.
Purpose of the Study:
- To optimize propidium monoazide (PMA) treatment conditions for accurate viable Escherichia coli quantification using PMA-qPCR.
- To evaluate the effectiveness of PMA-qPCR in differentiating live from heat-killed E. coli in cell suspensions and food matrices.
- To assess the applicability of PMA-qPCR for enumerating VBNC E. coli cells in food samples.
Main Methods:
- Systematic evaluation of different PMA concentrations (20-80 μM), light exposure times (5-30 min), and light intensities (500 W, 650 W).
- Application of optimized PMA-qPCR method to quantify live and heat-killed E. coli in cell suspensions and oyster matrices.
- Comparison of PMA-qPCR results with traditional plate counts and standard qPCR.
Main Results:
- Optimal PMA treatment conditions were determined as 50 μM PMA for 15 min under 650 W light intensity.
- PMA-qPCR significantly inhibited the amplification of DNA from heat-killed E. coli at concentrations ≤5 Log CFU/mL in cell suspensions.
- PMA-qPCR successfully enumerated VBNC E. coli in oyster samples and showed promise for accurate bacterial quantification in food matrices.
Conclusions:
- Optimized PMA-qPCR offers a reliable method for quantifying viable Escherichia coli by effectively excluding DNA from dead cells.
- The method demonstrates applicability for detecting VBNC E. coli in food, improving upon culture-dependent techniques.
- While PMA-qPCR shows significant promise for food safety, further validation across diverse food matrices is recommended to mitigate potential false-positive results.
