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Abstract
Magnetic separation is an efficient method for target enrichment and elimination of inhibitors in the molecular detection systems for foodborne pathogens. In this study, we prepared magnetic capture probes by modifying oligonucleotides complementary to target sequences on the surface of amino-modified silica-coated magnetic nanoparticles and optimized the conditions and parameters of probe synthesis and hybridization. We innovatively put the complexes of magnetic capture probes and target sequences into qPCR without any need for denaturation and purification steps. This strategy can reduce manual steps and save time. We used the magnetic capture probes to separate invA mRNA from Salmonella in artificially contaminated milk samples. The detection sensitivity was 104 CFU/ml, which could be increased to 10 CFU/ml after a 12 h enrichment step. The developed method is robust enough to detect live bacteria in a complex environmental matrix.
Highlights
Magnetic nanoparticles and especially immunomagnetic nanoparticles have been widely used for foodborne pathogen detection (Escalante-Maldonado et al, 2015; Sun et al, 2015; Hwang et al, 2016; Luciani et al, 2016; Wang et al, 2016)
Magnetic nanoparticles labeled with complementary sequences were used to capture target DNA sequences containing barcodes of Listeria monocytogenes followed by amplification and identification by polymerase chain reaction
The results showed that when the amount of magnetic capture probes added in Quantitative PCR (qPCR) was under 60 μg, the amplification was not affected
Summary
Magnetic nanoparticles and especially immunomagnetic nanoparticles have been widely used for foodborne pathogen detection (Escalante-Maldonado et al, 2015; Sun et al, 2015; Hwang et al, 2016; Luciani et al, 2016; Wang et al, 2016). The labeled antibody is a key point for a successful immunomagnetic detection method, and a limiting step is the quality of the anti-pathogen antibody used. Developments in molecular biology, genomics, and bioinformatics enable specific nucleotide sequences to be developed as barcodes for detection of target pathogens. The nucleotide sequence adjacent to the pathogen-specific barcode can be used as a medium to purify the detection sequence. Magnetic nanoparticles labeled with complementary sequences were used to capture target DNA sequences containing barcodes of Listeria monocytogenes followed by amplification and identification by polymerase chain reaction
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