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A Rapid and Sensitive Europium Nanoparticle-Based Lateral Flow Immunoassay Combined with Recombinase Polymerase Amplification for Simultaneous Detection of Three Food-Borne Pathogens.

ABSTRACT: Food-borne pathogens have become an important public threat to human health. There are many kinds of pathogenic bacteria in food consumed daily. A rapid and sensitive testing method for multiple food-borne pathogens is essential. Europium nanoparticles (EuNPs) are used as fluorescent probes in lateral flow immunoassays (LFIAs) to improve sensitivity. Here, recombinase polymerase amplification (RPA) combined with fluorescent LFIA was established for the simultaneous and quantitative detection of Listeria monocytogenes, Vibrio parahaemolyticus, and Escherichia coliO157:H7. In this work, the entire experimental process could be completed in 20 min at 37 °C. The limits of detection (LODs) of EuNP-based LFIA-RPA were 9.0 colony-forming units (CFU)/mL for Listeria monocytogenes, 7.0 CFU/mL for Vibrio parahaemolyticus, and 4.0 CFU/mL for Escherichia coliO157:H7. No cross-reaction could be observed in 22 bacterial strains. The fluorescent LFIA-RPA assay exhibits high sensitivity and good specificity. Moreover, the average recovery of the three food-borne pathogens spiked in food samples was 90.9-114.2%. The experiments indicate the accuracy and reliability of the multiple fluorescent test strips. Our developed EuNP-based LFIA-RPA assay is a promising analytical tool for the rapid and simultaneous detection of multiple low concentrations of food-borne pathogens.

SUBMITTER: Chen K

PROVIDER: S-EPMC8123443 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:A fluorescent immunoassay based on europium nanoparticles (EuNPs-FIA) was developed for the simultaneous detection of antibiotic residues, solving the problems of single target detection and low sensitivity of traditional immunoassay methods. In the EuNPs-FIA, EuNPs were used as indictive probes by binding to anti-tetracyclines monoclonal antibodies (anti-TCs mAb), anti-sulphonamides monoclonal antibodies (anti-SAs mAb) and anti-fluoroquinolones monoclonal antibodies (anti-FQs mAb), respectively. Different artificial antigens were assigned to different regions of the nitrocellulose membrane as capture reagents. The EuNPs-FIA allowed for the simultaneous detection of three classes of antibiotics (tetracyclines, fluoroquinolones and sulphonamides) within 15 min. It enabled both the qualitative determination with the naked eye under UV light and the quantitative detection of target antibiotics by scanning the fluorescence intensity of the detection probes on the corresponding detection lines. For qualitative analysis, the cut-off values for tetracyclines (TCs), fluoroquinolones (FQs) and sulphonamides (SAs) were 3.2 ng/ml, 2.4 ng/ml and 4.0 ng/ml, respectively, which were much lower than the maximum residue limit in food. For quantitative analysis, these ranged from 0.06 to 6.85 ng/ml for TCs, 0.03-5.14 ng/ml for FQs, and 0.04-4.40 ng/ml for SAs. The linear correlation coefficients were higher than 0.97. The mean spiked recoveries ranged from 92.1 to 106.2% with relative standard deviations less than 8.75%. Among them, the three monoclonal antibodies could recognize four types of TCs, seven types of FQs and 13 types of SAs, respectively, and the detection range could cover 24 antibiotic residues with different structural formulations. The results of the detection of antibiotic residues in real samples using this method were highly correlated with those of high performance liquid chromatography (R 2 > 0.98). The accuracy and precision of the EuNPs-FIA also met the requirements for quantitative analysis. These results suggested that this multiplex immunoassay method was a promising method for rapid screening of three families of antibiotic residues.

Project description:Melioidosis is a severe infectious disease caused by gram-negative, facultative intracellular pathogen Burkholderia pseudomallei (B. pseudomallei). Although cases are increasing reported from other parts of the world, it is an illness of tropical and subtropical climates primarily found in southeast Asia and northern Australia. Because of a 40% mortality rate, this life-threatening disease poses a public health risk in endemic area. Early detection of B. pseudomallei infection is vital for prognosis of a melioidosis patient. In this study, a novel isothermal recombinase polymerase amplification combined with lateral flow dipstick (LF-RPA) assay was established for rapid detection of B. pseudomallei. A set of primer-probe targeting orf2 gene within the putative type III secretion system (T3SS) cluster genes was generated and parameters for the LF-RPA assay were optimized. Result can be easy visualized in 30 minutes with the limit of detection (LOD) as low as 20 femtogram (fg) (ca. 25.6 copies) of B. pseudomallei genomic DNA without a specific equipment. The assay is highly specific as no cross amplification was observed with Burkholderia mallei, members of the Burkholderia cepacia-complex and 35 non-B. pseudomallei bacteria species. Moreover, isolates from patients in Hainan (N = 19), Guangdong (N = 1), Guangxi (N = 3) province of China as well as in Australia (N = 3) and Thailand (N = 1) were retrospectively confirmed by the newly developed method. LODs for B. pseudomallei-spiked soil and blood samples were 2.1×103 CFU/g and 4.2×103 CFU/ml respectively. The sensitivity of the LF-RPA assay was comparable to TaqMan Real-Time PCR (TaqMan PCR). In addition, the LF-RPA assay exhibited a better tolerance to inhibitors in blood than TaqMan PCR. Our results showed that the LF-RPA assay is an alternative to existing PCR-based methods for detection of B. pseudomallei with a potentiality of early accurate diagnosis of melioidosis at point of care or in-field use.

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A Rapid and Sensitive Europium Nanoparticle-Based Lateral Flow Immunoassay Combined with Recombinase Polymerase Amplification for Simultaneous Detection of Three Food-Borne Pathogens.

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