Polyamidoamine (PAMAM) dendrimer-mediated biotin amplified immunomagnetic separation method coupled with flow cytometry for viable Listeria monocytogenes detection

Abstract

In this study, we developed a PAMAM dendrimer-mediated biotin amplified magnetic separation method that was coupled with flow cytometry (FCM) for viable L. monocytogenes detection. PAMAM dendrimer-mediated biotin amplified magnetic separation strategy isolated more than 89.15%±1.75% L. monocytogenes both in PBS solutions and in spiked lettuce samples at bacterial concentration below 104 CFU/mL. Propidium monoazide (PMA) treatment prior to PCR amplification eliminated the false-positive results from dead bacteria and detected viable L. monocytogenes sensitively and specifically. In this assay, a pair of specific primers was synthesized for the hly gene of L. monocytogenes that was modified with biotin and FAM (FITC) respectively. After PCR amplification, biotin and FAM (FITC) labeled amplicons were immobilized on the streptavidin coated magnetic microbeads, and the mean fluorescence intensity (MFI) of the microbeads was measured with flow cytometer. Combined PAMAM dendrimer-mediated biotin amplified magnetic separation with FCM assay for viable L. monocytogenes detection, and gave a limit of detection (LOD) as low as 3.5×101 CFU/mL in PBS and 3.5×102 CFU/g in spiked lettuce samples. Moreover, the method developed exhibited excellent specificity. Therefore, PAMAM dendrimer-mediated biotin amplified magnetic separation method coupled with FCM assay is a highly promising approach for L. monocytogenes detection.