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

Abstract

Pathogenic Escherichia coli 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 E. coli 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 E. coli 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.

Keywords

• quantitative PCR
• propidium monoazide (PMA)
• lactic acid
• detection of viable pathogens
• food matrix