Sensitive and rapid detection of E. coli O157:H7 elimination of false-positives using PMAxx-IMS-enhanced real-time PCR.

Abstract

Molecular diagnostic methods cannot distinguish dead bacteria, which can cause false-positive results. Furthermore, dead bacterial DNA can remain in food interminably, which can cause false-positive results unrelated to actual infection risk. Therefore, a method for detecting only viable bacteria that eliminates false-positive results due to dead bacteria must be developed. We optimized the improved propidium monoazide (PMAxx) dye to selectively bind to DNA via penetration into the damaged cell membranes of dead bacteria, thereby effectively blocking the amplification of dead bacterial DNA. Subsequently, the residual PMAxx and food matrix were effectively removed by immunomagnetic separation (IMS). Thus, the detection sensitivity improved through the separation and concentration of bacteria. In salad samples spiked with 105 CFU/g of dead bacteria, conventional qPCR detected signals even at 100 CFU/g of viable bacteria, leading to a false-positive result. In contrast, the PMAxx-IMS method eliminated these false-positive results and demonstrated specific detection of viable bacteria only at ≥104 CFU/g. This study is the first to combine improved PMAxx treatment and IMS to effectively eliminate false-positive results caused by dead bacteria while enhancing detection sensitivity. Consequently, the PMAxx-IMS based detection method significantly reduces false-positives due to the amplification of dead bacterial DNA and enables a more accurate quantification of viable pathogens. This approach improves the reliability of molecular diagnostics in food microbial safety assessments and offers rapid and sensitive detection of various foodborne pathogens across diverse food matrices and contamination levels.