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Published on: May 20, 2020
Propidium monoazide for viable Salmonella enterica detection by PCR and LAMP assays in comparison to RNA-based
Chayapa Techathuvanan1, Doris Helen D'Souza1
1Department of Food Science, University of Tennessee, 2605 River Drive, Knoxville, TN, 37996-4591, U.S.A.
Abstract:
Rapid and sensitive detection of live/infectious foodborne pathogens is urgently needed in order to prevent outbreaks and food recalls. This study aimed to (1) evaluate the incorporation of propidium monoazide (PMA) into PCR or LAMP assays to selectively detect viable Salmonella Enteritidis following sublethal heat or UV treatment, and autoclave sterilization; and (2) compare the detection of PMA-PCR and PMA-LAMP to DNA-based PCR and LAMP (without PMA), RNA-based RT-PCR and RT-LAMP, and culture-based methods. Nucleic acids (DNA or RNA) from 1-mL S. Enteritidis samples were used for PCR, RT-PCR, LAMP, and RT-LAMP assays. Serially diluted samples were plated on Xylose Lysine Tergitol-4 agar for cultural enumeration. Comparable detection of overnight cultured S. Enteritidis was obtained by PMA-PCR, PCR, and RT-PCR, though 1 to 2 log less sensitive than cultural assays. PMA-LAMP and RT-LAMP showed similar detection of overnight cultures, being 1 to 2 log less sensitive than the LAMP assay, and ∼4 log less than culture-based detection. Autoclaved S. Enteritidis did not test positive by RNA-based methods or PMA-PCR, but PMA-LAMP showed detection of 1 log CFU/mL. PMA-PCR and RT-PCR showed comparable detection of sublethal heat-treated cells to cultural assays, while PMA-LAMP showed 1 to 2 log less detection. Our results suggest that PMA-PCR and PMA-LAMP assays are not suitable for selective viable cell detection after UV treatment. While PMA-LAMP assay needs optimization, PMA-PCR shows promise for live/viable S. Enteritidis detection. PMA-PCR shows potential for routine testing in the food industry with results within 1-day, albeit depending on the inactivation method employed.
Insights
Propidium monoazide (PMA)-PCR shows promise for detecting viable Salmonella Enteritidis in food, offering rapid results for industry testing. However, PMA-PCR and PMA-LAMP require further optimization for reliable detection after UV treatment.
Area of Science:
- Food Microbiology
- Molecular Biology
- Food Safety
Background:
- Rapid detection of viable foodborne pathogens like Salmonella Enteritidis is crucial for preventing outbreaks.
- Existing methods for detecting viable bacteria have limitations in speed and sensitivity.
Purpose of the Study:
- To evaluate propidium monoazide (PMA) incorporation into PCR and LAMP assays for selective detection of viable Salmonella Enteritidis.
- To compare PMA-PCR and PMA-LAMP with DNA-based, RNA-based, and culture-based methods.
Main Methods:
- Propidium monoazide (PMA) was used with PCR and LAMP assays.
- Nucleic acids (DNA/RNA) were extracted from treated S. Enteritidis samples.
- Assays included PMA-PCR, PMA-LAMP, DNA-PCR, DNA-LAMP, RT-PCR, RT-LAMP, and culture methods.
- Bacterial viability was assessed after heat, UV, or autoclave treatments.
Main Results:
- PMA-PCR and RT-PCR showed comparable detection of heat-treated cells to culture methods.
- PMA-PCR demonstrated potential for detecting viable S. Enteritidis after certain inactivation methods.
- Both PMA-PCR and PMA-LAMP were unsuitable for viable cell detection after UV treatment.
- PMA-LAMP requires further optimization for viable cell detection.
Conclusions:
- PMA-PCR shows promise as a rapid method for routine testing of viable Salmonella Enteritidis in the food industry.
- Further optimization is needed for PMA-LAMP assays.
- The effectiveness of PMA-based assays is dependent on the specific inactivation method used for the bacteria.
