Rapid and accurate detection for Listeria monocytogenes in milk using ampicillin-mediated magnetic separation coupled with quantitative real-time PCR

Abstract

Listeria monocytogenes (L. monocytogenes) is a typical food-borne pathogen which poses a serious threat to global public health. Achieving rapid and sensitive detection of pathogens in food is critical to solve food safety problems. In this work, ampicillin functionalized magnetic beads (Amp-MBs) were synthesized and used as a capture probe for detection of L. monocytogenes in milk sample combined with quantitative real-time polymerase chain reaction (qPCR) with high sensitivity and specificity. Amp, as a broad spectrum antibiotic was selected to identify bacteria of the Listeria spp. powerfully, and the hly as a specific gene of L. monocytogenes was selected to establish qPCR for accurate detection. At optimal conditions, the capture efficiency of Amp-MBs for L. monocytogenes (101-106 CFU/mL) and other bacteria of Listeria spp. (105 CFU/mL) was higher than 90 %. The limit of detection of Amp-MBs-qPCR strategy for L. monocytogenes was 101 and 102 CFU/mL in PBS and artificially contaminated milk within 2.5 h, respectively. And this strategy could detect L. monocytogenes from high concentration Listeria spp. with ascendant specificity. These results demonstrated that the Amp-MBs-qPCR strategy was successfully established and applied to the specific detection of L. monocytogenes in milk samples.