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.

Using Cy5-dUTP labelling of RPA-amplicons with downstream microarray analysis for the detection of antibiotic resistance genes

“…To date, RPA-based assays and devices capable for point of care applications (such as lateral flow assays) use pre-labelled primer 11 , 16 . For the combination of NAATs with the microarray technology and a possible detection of hundreds of target sequences with equally amounts of primer sets, it is in our opinion more useful to have a static labelling system.…”

“…The combination of RPA with various subsequent detection methods is very easy and enables a broad range of applications 3 . The most commonly used one is the combination with some kind of lateral flow assays 10 , 11 . More than 40% of the published results in 2020 used lateral flow based detection of RPA amplicons.…”

“…Using techniques like lateral flow or quantitative assays as well as microarrays, labelling of the amplicons are essential for the detection. This is commonly achieved by pre-labelled oligonucleotides 11 , 16 .…”

Latest Trends in Lateral Flow Immunoassay (LFIA) Detection Labels and Conjugation Process

“… Chen K. et al (2021) performed a rapid quantitative assay based on the recombinase polymerase amplification technique in combination with a lateral flow immunoassay for the detection of Listeria monocytogenes , Vibrio parahaemolyticus , and Escherichia coli using carboxylic Europium nanoparticles (EuNPs) as signal molecules. The conjugation protocol of EuNPs with anti-digoxin monoclonal antibody was as follows: activation of 2 mg carboxylic EuNPs dissolved in 800 µl 2-(N-morpholino)-ethanesulfonic acid (0.05 M, pH 8.2) with 30 µl EDC by slow shaking for 30 min, then the solution was centrifuged at 12,000 rpm for 25 min.…”

Latex Microsphere-Based Bicolor Immunochromatography for Qualitative Detection of Neutralizing Antibody against SARS-CoV-2

“…A lateral flow immunoassay (LFIA), which combines chromatography technology with conventional immunoassay and nanomaterials, is considered the most attractive point-of-care testing device, because it exhibits several advantages, including simple technical requirements, rapid detection capability, portability, affordability, high detection accuracy, and high efficiency [ 25 ]. LFIAs have been widely applied in detecting pesticide [ 26 , 27 ] and veterinary drugs residues [ 28 , 29 ], toxicants [ 30 , 31 , 32 ], pathogens [ 32 , 33 ], various diseases biomarkers, as well as SARS-CoV-2 antigen/antibody [ 34 , 35 ]. Usually, the results of a LFIA are presented with the test and control lines showing the same color, resulting in it not being easy for users to discern the locations of test and control zones.…”