Comparing two magnetic separation pretreatment methods for molecular detection using (q)PCR assays targeting Staphylococcus aureus in milk as models

Abstract

Magnetic separation presents significant potential for culture-independent detection of foodborne pathogens in food samples. In this study, we compared two magnetic separation pretreatment strategies for molecular detection using (q)PCR assays targeting Staphylococcus aureus in milk as models. The first approach employed amino-modified silica magnetic particles (ASMPs) for DNA separation, while the second utilized pig IgG-labeled magnetic beads (IgG-MBs) for cell separation. In the ASMPs-based DNA separation strategy, a sensitivity of 36 CFU/mL was achieved when ASMPs-DNA complexes were employed as templates for PCR analysis. To mitigate noise signals originating from ASMPs during qPCR, DNA on the surface of ASMPs was replaced with dNTP, resulting in a sensitivity of 1.6 × 103 CFU/mL. The IgG-MBs-based cell separation approach yielded sensitivities of 3.6 × 104 CFU/mL for PCR and 1.6 × 103 CFU/mL for qPCR following DNA isolation from the bacteria-IgG-MBs complexes. Both methods exhibited exceptional specificity and robustness against background bacteria interference. However, neither approach effectively discriminated between live and dead bacteria. In comparison, the ASMPs-based DNA separation strategy exhibited superior potential especially when ASMPs do not influence the detection system. This research contributes to the optimization of magnetic separation pretreatment strategies for the molecular detection of foodborne pathogens, highlighting the potential of culture-independent detection methods.