Biotin exposure-based immunomagnetic separation coupled with sodium dodecyl sulfate, propidium monoazide, and multiplex real-time PCR for rapid detection of viable Salmonella Typhimurium, Staphylococcus aureus, and Listeria monocytogenes in milk.

Xiuquan Shi, Liang Yu, Cui Lin, Ke Li, Jihua Chen, Hong Qin

Journal of dairy science 2021 Jun

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In this study, we established a rapid and sensitive method for the detection of viable Salmonella Typhimurium, Staphylococcus aureus, and Listeria monocytogenes in milk using biotin-exposure-based immunomagnetic separation (IMS) combined with sodium dodecyl sulfate (SDS), propidium monoazide (PMA), and multiplex real-time PCR (mRT-PCR). We used IMS to lessen the assay time for isolation of target bacteria. We then optimized the coupling conditions and immunomagnetic capture process. The immunoreaction and incubation times for 5 μg of mAb coupled with 500 μg of streptavidin-functionalized magnetic beads using a streptavidin-biotin system were 90 and 30 min, respectively. Treatment with SDS-PMA before mRT-PCR amplification eliminated false-positive outcomes from dead bacteria and identified viable target bacteria with good sensitivity and specificity. The limit of detection of IMS combined with the SDS-PMA-mRT-PCR assay for the detection of viable Salmonella Typhimurium, Staph. aureus, and L. monocytogenes in spiked milk matrix samples was 10 cfu/mL and remained significant even in the appearance of 106 cfu/mL of nontarget bacteria. The entire detection process was able to identify viable bacteria within 9 h. The combination of biotin-exposure-mediated IMS and SDS-PMA-mRT-PCR has potential value for the rapid and sensitive detection of foodborne pathogens. Copyright © 2021 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

Citation

Xiuquan Shi, Liang Yu, Cui Lin, Ke Li, Jihua Chen, Hong Qin. Biotin exposure-based immunomagnetic separation coupled with sodium dodecyl sulfate, propidium monoazide, and multiplex real-time PCR for rapid detection of viable Salmonella Typhimurium, Staphylococcus aureus, and Listeria monocytogenes in milk. Journal of dairy science. 2021 Jun;104(6):6588-6597