A risk-tiered framework for detecting viable but non-culturable foodborne pathogens: a critical synthesis from...

The viable but non-culturable (VBNC) state is a critical food safety blind spot. Major pathogens (E. coli O157:H7, S. enterica, L. monocytogenes, C. jejuni) enter a metabolically active, virulent dormancy under common processing stresses, evading culture-based detection. Here, viability is inferred operationally from membrane integrity, metabolic activity, or resuscitation capacity. While the direct disease burden from VBNC cells remains unquantified, their invisibility creates a systematic risk-assessment gap and may underlie unexplained outbreaks. This critical review synthesizes current and emerging VBNC detection technologies, evaluating performance against real-world food matrix challenges (fats, proteins, polyphenols, high salt). We derive an evidence-based, risk-tiered framework through comparative analysis that weighs operational objectives against technology capabilities and matrix compatibilities, operationalized via a decision flowchart and Technology Readiness Level (TRL) matrix. No single method resolves the detection gap; PMAxx-RPA-LFA is optimal for rapid point-of-need screening (Tier 1, approximately 50-60 min), PMA-dPCR for regulatory confirmation and absolute quantification (Tier 2), AI-enhanced hyperspectral microscopy or targeted metatranscriptomics for research and outbreak investigation (Tier 3), and integrated microfluidic phage-CRISPR-AI platforms as the Tier 4 horizon. Three challenges are identified: absent standardized VBNC induction protocols, lack of validated VBNC-specific biomarkers, and the need to link detection signals to public health risk via quantitative microbial risk assessment (QMRA). A 5-year roadmap is proposed to guide proactive food safety management.