Improving the Efficiency of Viability-qPCR with Lactic Acid Enhancer for the Selective Detection of Live Pathogens in Foods

Pathogenic are the most prevalent foodborne bacteria, and their accurate detection in food samples is critical for ensuring food safety. Therefore, a quick technique named viability-qPCR (v-qPCR), which is based on the ability of a selective dye, such as propidium monoazide (PMA), to differentiate b...

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Bibliographic Details
Published in:Foods 2024-04, Vol.13 (7), p.1021
Main Authors: Dinu, Laura-Dorina, Al-Zaidi, Quthama Jasim, Matache, Adelina Georgiana, Matei, Florentina
Format: Article
Language:English
Subjects:
Acids
Bacteria
Cell differentiation
detection of viable pathogens
Dyes
E coli
Efficiency
Escherichia coli
Food
food matrix
Food safety
Foodborne pathogens
Gram-negative bacteria
Heat
Lactic acid
Milk
Molecular biology
Pathogenic microorganisms
Pathogens
Permeability
propidium monoazide (PMA)
quantitative PCR
Safety and security measures
Salmonella
Surfactants
Toxicity
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Summary:Pathogenic are the most prevalent foodborne bacteria, and their accurate detection in food samples is critical for ensuring food safety. Therefore, a quick technique named viability-qPCR (v-qPCR), which is based on the ability of a selective dye, such as propidium monoazide (PMA), to differentiate between alive and dead cells, has been developed. Despite diverse, successful applications, v-qPCR is impaired by some practical limitations, including the ability of PMA to penetrate the outer membrane of dead Gram-negative bacteria. The objective of this study is to evaluate the ability of lactic acid (LA) to improve PMA penetration and, thus, the efficiency of v-qPCR in detecting the live fraction of pathogens. The pre-treatment of ATCC 8739 cells with 10 mM LA greatly increased PMA penetration into dead cells compared to conventional PMA-qPCR assay, avoiding false positive results. The limit of detection when using LA-PMA qPCR is 1% viable cells in a mixture of dead and alive cells. The optimized LA-PMA qPCR method was reliably able to detect log 2 CFU/mL culturable in milk spiked with viable and non-viable bacteria. Lactic acid is cheap, has low toxicity, and can be used to improve the efficiency of the v-qPCR assay, which is economically interesting for larger-scale pathogen detection applications intended for food matrices.
ISSN:2304-8158
2304-8158
DOI:10.3390/foods13071021