Enhanced Detection of Viable Escherichia coli O157:H7 in Romaine Lettuce Wash Water Using On-Filter Propidium Monoazide-Quantitative PCR

Abstract

Accurate detection of viable Escherichia coli O157:H7 in fresh produce wash water is critical for ensuring food safety and mitigating foodborne illnesses. This study evaluated an optimized on-filter propidium monoazide (PMA)-quantitative PCR (qPCR) method for detecting viable E. coli O157:H7 in romaine lettuce wash water, involving PMA pretreatment on a filter to block DNA amplification from dead cells. The method consistently detected viable cells across chemical oxygen demand (COD) levels of 1000 and 200 mg O2/L, with no significant differences (p > 0.05), indicating its tolerance to organic matter interference. Optimization experiments identified 10 µM PMA with a 10 min exposure time as the most effective pretreatment, achieving efficient inhibition of DNA from dead cells while preserving viable cell integrity. The limit of detection (LOD) was 1.3 CFU/mL, confirming its suitability for detecting low bacterial loads. Performance evaluations revealed that PMA-qPCR was accurate at viable-to-dead cell ratios of 1:10 or higher but became less reliable when dead cells outnumbered viable cells by a factor of 10 or more. The study demonstrates the potential of on-filter PMA-qPCR for routine food safety monitoring protocols in the fresh produce industry, while highlighting the critical role of viable-to-dead cell ratios in ensuring accurate detection, particularly in challenging samples with high dead cell loads.