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Biotin-exposure-based immunomagnetic separation coupled with nucleic acid lateral flow biosensor for visibly detecting viable Listeria monocytogenes

Li, Fulai, Li, Fan, Luo, Dan, Lai, Weihua, Xiong, Yonghua, Xu, Hengyi
Analytica chimica acta 2018 v.1017 pp. 48-56
Listeria monocytogenes, analytical chemistry, antibodies, bacteria, biosensors, biotin, false positive results, food pathogens, immunomagnetic separation, infectious diseases, lettuce, magnetism, nanoparticles, nucleic acids, phosphates, polymerase chain reaction, propidium, public health, rapid methods, risk, screening, streptavidin
Infectious diseases caused by Listeria monocytogenes pose a great threat to public health worldwide. Therefore, a rapid and efficient method for L. monocytogenes detection is needed. In this study, a biotin-exposure-based immunomagnetic separation (IMS) method was developed. That is, biotinylated antibody was first targeted to L. monocytogenes. Then, streptavidin-functionalized magnetic nanoparticles were added and anchored onto L. monocytogenes cells indirectly through the strong noncovalent interaction between streptavidin and biotin. Biotin-exposure-based IMS exhibited an excellent capability to enrich L. monocytogenes. Specifically, more than 90% of L. monocytogenes was captured when the bacterial concentration was lower than 10⁴ colony-forming units (CFU)/mL. Importantly, the antibody dosage was reduced by 10 times of that in our previous study, which used antibody direct-conjugated magnetic nanoparticles. Propidium monoazide (PMA) treatment prior to PCR amplification could eliminate the false-positive results from dead bacteria and detected viable L. monocytogenes sensitively and specifically. For viable L.monocytogenes detection, enriched L. monocytogenes was treated with PMA prior to asymmetric PCR amplification. The detection limits of the combined IMS with nucleic acid lateral flow (NALF) biosensor for viable L. monocytogenes detection were 3.5 × 10³ CFU/mL in phosphate buffer solution and 3.5 × 10⁴ CFU/g in lettuce samples. The whole assay process of recognizing viable L. monocytogenes was completed within 6 h. The proposed biotin-exposure-mediated IMS combined with a disposable NALF biosensor platform posed no health risk to the end user, and possessed potential applications in the rapid screening and identification of foodborne pathogens.