Nucleic Acid Lateral Flow Assay Research Articles

MNAzyme-Assisted Nucleic Acid Lateral Flow Assay for Cost-Effective, On-Site Mercury Detection.

Mercury ions (Hg2+) are toxic heavy metals present in the environment that pose significant health risks. An advanced detection system could allow for a prompt response and alleviate serious damage to humans. In this study, we developed a cost-effective, on-site detection method for Hg2+ using a multicomponent nucleic acid enzyme (MNAzyme)-assisted nucleic acid lateral flow assay (NALFA). The MNAzyme, which was engineered to contain thymine-thymine mismatches, is responsive only to the presence of Hg2+ and exerts efficient cleavage activity on substrates that can be captured by the NALFA strip, and thus the proposed system enables the visual detection of Hg2+ in the NALFA strip. Our assay demonstrated sufficient detection sensitivity and specificity to meet the WHO standards, offering a good practical alternative for rapid environmental and public health monitoring.

Janus Fe3O4-Au@Pt nanozymes based lateral flow assay for enhanced-sensitive colorimetric detection of influenza A virus

The traditional colloidal gold-based lateral flow assay (LFA) is widely applied in point-of-care testing (POCT) for pathogens. However, its disadvantage of low sensitivity greatly limits its effectiveness in early detection. In this work, we developed an enhanced-sensitive colorimetric nucleic acid lateral flow assay (NALFA) based on Janus Fe3O4-Au@Pt nanoparticles (Fe3O4-Au@Pt NPs) for H1N1 influenza virus detection. Dumbbell-like heterodimer Fe3O4-Au@Pt NPs were prepared by a seed-mediated method, and their unique Janus structure endowed them with both good plasmonic property and high peroxidase-like activity. With the Fe3O4-Au@Pt NPs as NALFA signal labels, qualitative detection was achieved by observing the color change with the naked eyes, and quantitative results were obtained by measuring the gray values on test strips. Due to the strong peroxidase-like activity of Fe3O4-Au@Pt NPs, they could quickly catalyze 3,3′,5,5′-tetramethylbenzidine (TMB) to oxidize to the dark blue chelate (oxTMB), thus resulting in greatly enhanced color on the strips. The limit of detection (LOD) of NALFA reached as low as 50 pM, which was 20-fold lower than that of before catalysis. Therefore, the constructed Janus Fe3O4-Au@Pt NPs could effectively improve the performance of traditional colloidal gold LFA and provide a potential tool for the development of sensitive sensing platforms.

Self-assembly of DNA-gold nanoaggregate for visual detection of thymidine kinase 1 (TK1) mRNA via lateral flow assay.

Nucleic acid lateral flow assay (NALFA) with gold nanoparticles (AuNPs) as colorimetric probes have been extensively adopted for point-of-care testing (POCT). However, the sensitivity of NALFA still needs to be improved. Herein, DNA-gold nanoaggregate (DNA-AuNA) was assembled as a signal amplification probe of NALFA for sensitive detection of tumor marker TK1 mRNA. Four functional oligonucleotides with complementary pairs were assembled to form DNA-AuNA that coupled more AuNPs to improve sensitivity. Thus, the limit of detection (LOD) was 0.36pM, which is lower than that of conventional AuNPs-based NALFA. Moreover, the bioassay showed good reproducibility, stability, and specificity for detecting TK1 mRNA. The detection of TK1 mRNA in human serum was also satisfactory. Therefore, DNA-AuNA-based NALFA provides a sensitive method for portable detection of TK1 mRNA.

Isothermal Amplification and Hypersensitive Fluorescence Dual-Enhancement Nucleic Acid Lateral Flow Assay for Rapid Detection of Acinetobacter baumannii and Its Drug Resistance.

Acinetobacter baumannii (A. baumannii) is among the main pathogens that cause nosocomial infections. The ability to rapidly and accurately detect A. baumannii and its drug resistance is essential for blocking secondary infections and guiding treatments. In this study, we reported a nucleic acid fluorescent lateral flow assay (NFLFA) to identify A. baumannii and carbapenem-resistant A. baumannii (CRAB) in a rapid and quantitative manner by integrating loop-mediated isothermal amplification (LAMP) and silica-based multilayered quantum dot nanobead tag (Si@MQB). First, a rapid LAMP system was established and optimised to support the effective amplification of two bacterial genes in 35 min. Then, the antibody-modified Si@MQB was introduced to capture the two kinds of amplified DNA sequences and simultaneously detect them on two test lines of a LFA strip, which greatly improved the detection sensitivity and stability of the commonly used AuNP-based nucleic acid LFA. With these strategies, the established LAMP-NFLFA achieved detection limits of 199 CFU/mL and 287 CFU/mL for the RecA (house-keeping gene) and blaOXA-23 (drug resistance gene) genes, respectively, within 43 min. Furthermore, the assay exhibited good repeatability and specificity for detecting target pathogens in real complex specimens and environments; thus, the proposed assay undoubtedly provides a promising and low-cost tool for the on-site monitoring of nosocomial infections.

Two-color lateral flow nucleic acid assay combined with double-tailed recombinase polymerase amplification for simultaneous detection of chicken and duck adulteration in mutton

We propose a visual strategy for simultaneous detection of chicken and duck ingredients in mutton by integration of a double tailed multiplex recombinase polymerase amplification (mRPA) assay with a multicolor nucleic acid hybridization lateral flow strip (mNAH-LFS) assay. Two colored gold nanoparticles (AuNPs), including gold nanospheres (red) and gold nanoflowers (blue), were synthesized and functionalized with the corresponding nucleic acid probe to target each analyte. The RPA product was analyzed by this two-colored mNAH-LFS assay which includes a red T1 line for chicken adulteration and a blue T2 line for duck adulteration. In this RPA reaction, special Spacer9 and tail nucleotide sequence-tagged primers (Tag+Spacer9+F-primer, Tag+Spacer9+R-primer) were used to obtain double-stranded amplicons with specific wobbled sequences that can directly bind with AuNPs-labeled probes (AuNSs-cT1 hybridized with T1, AuNFs-cT4 hybridized with T4) and solid-phase probes (biotin-cT2 hybridized with T2, biotin-cT3 hybridized with T3) on the mNAH-LFS producing a red or/and cyan color on the T lines. The detection limits of this mRPA-mNAH-LFS assay for chicken and duck were 0.01% and 0.01%, respectively. Finally, this mRPA-mNAH-LFS assay was successfully used to authenticate 59 commercial meat products. In conclusion, this mRPA-mNAH-LFS is a sensitive, accurate, and cost-effective approach for meat quality assurance and traceability.

A Molecular Lateral Flow Assay for SARS-CoV-2 Quantitative Detection.

Since the onset of the SARS-CoV-2 pandemic, several COVID-19 detection methods, both commercially available and in the lab, have been developed using different biomolecules as analytes and different detection and sampling methods with high analytical performance. Developing novel COVID-19 detection assays is an exciting research field, as rapid accurate diagnosis is a valuable tool to control the current pandemic, and also because the acquired knowledge can be deployed for facing future infectious outbreaks. We here developed a novel gold-nanoparticle-based nucleic acid lateral flow assay for the rapid, visual, and quantitative detection of SARS-CoV-2. Our method was based on the use of a DNA internal standard (competitor) for quantification and involved RT-PCR, the hybridization of biotinylated PCR products to specific oligonucleotide probes, and detection with a dual lateral flow assay using gold nanoparticles conjugated to an anti-biotin antibody as reporters. The developed test allowed for rapid detection by the naked eye and the simultaneous quantification of SARS-CoV-2 in nasopharyngeal swabs with high specificity, detectability, and repeatability. This novel molecular strip test for COVID-19 detection represents a simple, cost-effective, and accurate rapid test that is very promising to be used as a future diagnostic tool.

DNA barcode-based detection of exosomal microRNAs using nucleic acid lateral flow assays for the diagnosis of colorectal cancer.

Colorectal cancer (CRC) is the third most common cancer and the second leading cause of cancer-related deaths worldwide. The standard methods for diagnosing CRC, endoscopy and tissue biopsy, are invasive and time-consuming. Herein, we propose a novel method for the accurate and non-invasive diagnosis of CRC based on the analysis of exosomes that are circulating in biological fluids using a DNA barcode-based nucleic acid lateral flow assay (NALFA). Our technology combines reverse transcription using a stem-loop primer with DNA barcode-based NALFA. A colorimetric signal is generated only in the presence of the target exosomal miRNA, which can be determined even with the naked eye. The proposed system successfully detected miR-92a and miR-141, which are overexpressed in CRC exosomes. Moreover, when applied to plasma samples from CRC patients, our system simultaneously detected multiple markers in one strip. By combining these markers, we achieved high analytical performance with a sensitivity and a specificity of 95.24% and 100.0%, respectively, demonstrating that the proposed assay can be a simple diagnostic platform for the detection of exosomal miRNA.

Unreacted Labeled PCR Primers Inhibit the Signal in a Nucleic Acid Lateral Flow Assay as Elucidated by a Transport Reaction Model.

Factors that affect the performance of the nucleic acid lateral flow assay (NALFA) have not been well studied. In this work, we identify two important phenomena that negatively affect signal intensities during the detection of PCR products using NALFA: (i) the presence of unreacted PCR primers, and (ii) the presence of excess PCR amplicons. This is the first report that highlights the negative effect of unreacted PCR primers on NALFA. The negative effect of excess amplicons, while not explicitly reported for NALFAs, emanates from an identical phenomenon in lateral flow immunoassays known as the "hook effect". We show that the above effects may be alleviated by increasing the concentration of capture antibodies at the test line and the concentration of reporter moieties (gold nanoparticles). To demonstrate these, we utilized a PCR assay in which both primers were end-labeled, to generate dually end-labeled (bi-labeled) PCR amplicons of 230 bp length. To provide mechanistic understanding of these phenomena, we present the first transport-reaction model of NALFA, the results of which qualitatively matched all observed phenomena. Based on these results, we provide recommendations for the optimal design of PCR for NALFA detection.

Loop-mediated isothermal amplification-based nucleic acid lateral flow assay for the specific and multiplex detection of genetic markers

In this study, a loop-mediated isothermal amplification-based nucleic acid lateral flow assay (LAMP-NALFA) system was developed for the specific and multiplex detection of genetic markers at a low cost. In principle, the LAMP reaction was optimized to generate a single-stranded sequence in the LAMP product, which was designed to serve as a barcode sequence and to specifically bind to the DNA capture on a NALFA strip. As a target genetic marker, the Salmonella enterotoxin (stn) gene was chosen and determined down to 9 aM (∼5.44 copies/μL). Importantly, the proposed system clearly discriminated the specific target amplification products from non-specific amplification products resulting from primers or non-target nucleic acids, proving the high selectivity of the LAMP-NALFA system. Furthermore, the practical applicability of the system was demonstrated by detecting Salmonella bacteria in Luria-Bertani medium, drinking water, and eggshells, with a limit of detection of 1.6 CFU. Finally, two different bacteria (Salmonella and Staphylococcus) were simultaneously determined by the multiplex LAMP-NALFA system. It is expected that the LAMP-NALFA system could be used in a point-of-care setting for the detection of bacteria or viruses, consequently improving both individual and public health.

Affinity-Switchable Lateral Flow Assay.

Lateral flow assay (LFA) has been a valuable diagnostic tool in many important fields where rapid, simple, and on-site detection is required, for applications such as pregnancy tests and infectious disease prevention. Currently, two types of LFAs are available: lateral flow immunoassay (LFIA) and nucleic acid lateral flow assay (NALFA). Both are generally used for the testing of proteins and nucleic acids. However, enzyme activities and small molecules without the corresponding binding partner cannot be detected by the existing LFAs. In this paper, we introduce a LFA approach termed affinity-switchable lateral flow assay (ASLFA) to overcome the limitations. The detection principle is based on the switchable binding between the affinity-switchable biotin (ASB) probe and avidin protein. In the presence of the target molecule, the activated ASB probe would be captured by the avidin, thereby leaving a distinct test line on the membrane. The ASLFA concept was demonstrated by testing the F ion, NADH cofactor, and nitroreductase activity. Thus, this general ASLFA can be used for the rapid detection of molecules that cannot be accessed by the classical LFAs.

Comparative Study of Gold and Carbon Nanoparticles in Nucleic Acid Lateral Flow Assay.

A lateral flow assay (LFA) is a paper-based, point-of-need test designed to detect a specific analyte in complex samples in low-resource settings. Although LFA has been successfully used in different applications, its use is still limited when high sensitivity is required, especially in the diagnosis of an early-stage condition. The limit of detection (LOD) is clearly related to the signal-generating system used to achieve the visual readout, in many cases involving nanoparticles coupled to a biomolecule, which, when combined, provides sensitivity and specificity, respectively. While colloidal gold is currently the most-used label, other detection systems are being developed. Carbon nanoparticles (CNPs) demonstrate outstanding features to improve the sensitivity of this technology by producing an increased contrast in the paper background. Based on the necessity of sensitivity improvement, the aim of this work is a comparative study, in terms of analytical performance, between commercial streptavidin gold nanoparticles (streptAv-AuNPs) and avidin carbon nanoparticles (Av-CNPs) in a nucleic acid lateral flow assay. The visual LOD of the method was calculated by serial dilution of the DNA template, ranging from 0.0 to 7 pg μL−1/1.5 × 104 CFU mL−1). The LFA achieved visual detection of as low as 2.2 × 10−2 pg μL−1 using Av-CNPs and 8.4 × 10−2 pg μL−1 using streptAv-AuNPs. These LODs could be obtained without the assistance of any instrumentation. The results demonstrate that CNPs showed an increased sensitivity, achieving the nanomolar range even by visual inspection. Furthermore, CNPs are the cheapest labels, and the suspensions are very stable and easy to modify.

DNA immobilization and detection using DNAbinding proteins.

The immobilization of sensing bioreceptors is a critical feature affecting the final performance of a biosensor. For DNA detection, the (strept)avidin-biotin affinity interaction is often used for the immobilization of biotin-labeled oligonucleotides or PCR amplicons. Herein, DNAbinding proteins are proposed as alternative universal anchors for both DNA immobilization and detection, based on the strong and specific affinity interaction between certain DNAbinding proteins and their respective dsDNA binding sites. These binding sites can be incorporated in the target DNA molecule during synthesis and by PCR, eliminating the need for post-synthesis chemical modification and resulting in lower costs. When scCro DNAbinding protein was immobilized on microplates and nitrocellulose membrane, both ssDNA and dsDNA targets were successfully detected. The detection limits achieved were similar to those obtained with the streptavidin-biotin system. However, the scCro system resulted in higher signals while using less amount of protein. The adsorption properties of scCro were superior to streptavidin's, making scCro a viable alternative as an anchor biomolecule for the development of DNA assays and biosensors. Finally, a nucleic acid lateral flow assay based solely on two different DNAbinding proteins, scCro and dHP, was developed for the detection of a PCR amplicon. Overall, the proposed system appears to be very promising and with potential use for multiplex detection using various DNAbinding proteins with different sequence specificities. Further work is required to better understand the adsorption properties of these biomolecules on nitrocellulose, optimize the assays comprehensively, and achieve improved sensitivities.

Rapid on-site detection of Salmonella pullorum based on lateral flow nucleic acid assay combined with recombinase polymerase amplification reaction

Caused by Salmonella pullorum, pullorosis is a bacterial disease threatening the poultry industry and has been listed as the bacterial disease to be eliminated by the government. However, antibiotic treatment of pullorosis has become increasingly difficult, resulting in severe influences on the sustainable development of poultry. Abuse of antibiotics may cause global drug-resistant problems. Hence, early diagnosis of young chickens and accurate treatment of sick chickens are urgently needed. Traditional serotyping for Salmonella detection is costly and labor-intensive, whereas other commonly used plate agglutination test methods often cause physical injury of chickens. Therefore, a rapid and nondamaging detection method is of great significance for early diagnosis, which is the key step in accurate medication and elimination of pullorosis. In this study, we propose a novel lateral flow nucleic acid assay (LFNAA) system combining recombinase polymerase amplification (RPA) for the detection of S. pullorum. In this method, the DNA of S. pullorum strains was quickly amplified by RPA under 37°C, and then, the RPA products were added onto the LFNAA sample pad until the final results could be observed by naked eyes within 3 min. The proposed assay is fast and delivers visible results to naked eyes in field test. The limit of detection for genomic DNA was 5 × 10−3 ng/μL, indicating high sensitivity. In addition, the proposed LFNAA system is cost-effective, efficient, and nondamaging to chicks in the field test. This system provides technical support for early diagnosis of S. pullorum in the poultry and paves a way for future precision medicine to avoid the global drug-resistance issues.

Novel ssDNA Ligand Against Ovarian Cancer Biomarker CA125 With Promising Diagnostic Potential.

The in-vitro diagnostic industry is always striving to explore specific and high-affinity recognition entities, sensitive probes, and newer technology or platforms to develop disease detection methods with lower production and time costs and with minimum interference or variability. Aptamers suffice as a reliable recognition element by addressing the issues mentioned earlier. Hence, this work focuses on screening high-affinity ssDNA ligands to capture an exemplary biomarker CA125 using membrane-SELEX technology coupled with aptainformatics (translational bioinformatics using aptamers). The ssDNA ligands or aptamers have been screened and characterized extensively through an array of assays to ensure a valuable diagnostic potential with KD (dissociation constant) of 166 nM. The robustness of the selected aptamer ligand 2.26 and its complex with target CA125 is investigated in the presence of serum and extreme salt concentrations. Its diagnostic potential is convincingly demonstrated by running a competitive nucleic acid lateral flow assay at various sample concentrations. The ssDNA ligand reported in this manuscript holds immense potential in the detection and specific targeting of CA125 biomarker.

Clustered Regularly Interspaced Short Palindromic Repeats/Cas9-Mediated Lateral Flow Nucleic Acid Assay.

The lateral flow assay is one of the most convenient analytical techniques for analyzing the immune response, but its applicability to precise genetic analyses is limited by the false-positive signal and tedious and inefficient hybridization steps. Here, we introduce the CRISPR (clustered regularly interspaced short palindromic repeats) /Cas system into the lateral flow assay, termed CRISPR/Cas9-mediated lateral flow nucleic acid assay (CASLFA), to address such issues. In this study, CASLFA is utilized to identify Listeria monocytogenes, genetically modified organisms (GMOs), and African swine fever virus (ASFV) at a detection limit of hundreds of copies of genome samples with high specificity within 1 h. We further evaluated the performance of CASLFA in a nonlaboratory environment and successfully confirmed 27 ASFV-infected samples from 110 suspected swine serum samples, with an accuracy of 100% when compared to real-time PCR (RT-PCR) assay. CASLFA satisfies some of the characteristics of a next-generation molecular diagnostics tool due to its rapidity and accuracy, allowing for point-of-care use without the need for technical expertise and complex ancillary equipment. This method has great potential for gene analysis in resource-poor or nonlaboratory environments.

Chitosan-modified nitrocellulose membrane for paper-based point-of-care testing

Green/sustainable cellulose paper-based platforms at point-of-care testing have received much interest in biomedical fields at resource-limited settings. In such platforms, biomolecules would have to be immobilized onto paper substrate to achieve detection. Therefore, effective molecule immobilization is critical for the performance of paper-based testing, especially the detection sensitivity. Although various methods have been developed to improve the detection sensitivity, the immobilization capacity of biomolecules has not been considered yet. In this study, we developed a facile method for modifying paper materials by incorporating chitosan into NC membrane, which has positive effect on the pore size, porosity, and surface groups of NC membrane, hence, enhancing the immobilization capacity of biomolecules on paper substrate. We demonstrated the effectiveness of the above mentioned approach by using nucleic acid lateral flow assay (NALFAs) as a model paper-based platform; in comparison with the unmodified NALFAs, the results showed ten-fold increase in the detection sensitivity for HBV. We envision that this facile modification method will have a great potential for developing other highly sensitive paper-based detecting platforms.

A low-cost novel lateral flow nucleic acid assay (LFNAA) for yak milk authentication

A low-cost novel lateral flow nucleic acid assay (LFNAA) was developed for rapid and sensitive authentication of yak milk on-site. In this method, a special abasic spacer C3 and tail nucleotide sequence modified reverse primers were innovatively designed for rapid isothermal recombinant polymerase amplification (RPA) to obtain a double-stranded amplicon with a single-stranded DNA tail, which could directly bind to gold nanoparticle (AuNP)-labeled detection probes, and produce a sandwich complex to show a red color on the test line of the strip. Compared to conventional RPA-lateral flow assay based on expensive antibodies, the proposed method in this work demonstrated massive cost reduction, avoided time-consuming and laborious AuNP-antibody conjugation process. The LFNAA revealed high specificity and sensitivity, which allowed detecting as low as 5% of yak milk within 40 min. This LFNAA facilitated non-specialized operators to field determine the authenticity of yak milk by naked eyes, and could be readily applied to other nucleic acid detection such as food safety inspection and point-of-care medical tests.

Nucleic acid lateral flow assay with recombinase polymerase amplification: Solutions for highly sensitive detection of RNA virus

We propose nucleic acid lateral flow assay (LFA) coupled with reverse transcription recombinase polymerase amplification (RT-RPA) resulting from step-by-step multiparametric adjustments to both RT-RPA reactions and LFA interactions. The assay was realized for RNA virus detection using the example of potato virus X (PVX), a dangerous phytopathogen. The assay stages were adjusted for sensitive detection. (1) DNA target was designed and verified. A fragment (146 bp) of coat protein gene (gp5) and biotin-/fluorescein-labeled forward/reverse primers were chosen to produce target amplicons. (2) In a test strip, the construction advantage of the realization of the highest affinity interaction (biotin–streptavidin in our research) through gold nanoparticle conjugate (streptavidin immobilized on the GNP surface) was demonstrated. (3) RPA with reverse transcription was adjusted including primer concentration, order of components’ mixing, and reaction temperature. Due to the adjustments, the assay was able to detect 0.14 ng PVX per g potato leaves at 30 min. The PVX assay was 260 times more sensitive than conventional lateral flow assay based on antibodies and demonstrated the same sensitivity as PCR detection. The proposed adjustments are applicable for ultrasensitive and rapid detection of various RNA viruses.

Smartphone detection of antibiotic resistance using convective PCR and a lateral flow assay

Rapid and simple nucleic acid tests are critical for the emerging field of personalised medicine. In particular, rapid detection of antibiotic resistance in Staphylococcus aureus is crucial in initiating appropriate antibiotic treatment that can be used in routine clinical situations. Our aim was to develop a rapid detection method based on convective PCR (cPCR) in combination with a nucleic acid lateral flow assay. cPCR offers an alternative rapid amplification of nucleic acids in less than 30 min without the need for complex equipment. A low-cost cPCR device was developed with off-the shelf electronic components that allows amplification of nucleic acids without the need for electrical power. The multiplex cPCR was performed using fluorescein- and digoxigenin-modified primers targeting the femA and mecA genes to differentiate methicillin-sensitive and -resistant S. aureus in a single tube. The mecA gene is responsible for methicillin-resistance while the femA gene allows identification of the causative organism. The modified amplicons were analysed with a duplex lateral flow assay using quantum dot-labelled reporter probes and the fluorescent signal was acquired via a smartphone camera. The utility of our diagnostic system was demonstrated by detecting clinical samples of MRSA with a detection limit of 4.7 × 103 copies of DNA. The smartphone system developed may find facile applications outside centralised laboratories, such as in doctor’s offices and at the bed-side of patients.

Ultra-Fast On-Site Molecular Detection of Foodborne Pathogens Using a Combination of Convection Polymerase Chain Reaction and Nucleic Acid Lateral Flow Immunoassay.

Rapid detection and timely treatment of diseases caused by foodborne pathogens is important for improving the curative efficiency and preventing the spread of disease. In this study, we developed an assay utilizing a recently introduced ultra-fast convection polymerase chain reaction (cPCR) in conjunction with a simple nucleic acid lateral flow (NALF) immunoassay for ultra-fast on-site molecular detection of foodborne pathogens. Two Salmonella enterica serovars, Salmonella Enteritidis and Salmonella Typhimurium, and Escherichia coli O157:H7 were used as the target pathogens. We confirmed the specific amplification of the target species with specifically designed modified primer sets for cPCR in singleplex and duplex modes. After cPCR amplification, we compared the detection specificity and sensitivity using agarose gel electrophoresis and NALF assays with one or two test lines. The cPCR amplicons were readily and sensitively detected using the NALF assay, and the sensitivity was comparable with that of agarose gel electrophoresis. To confirm the application of the assay in real-life samples, the assay was used to test artificially contaminated milk. Without sample pre-enrichment, the limit of detection (LOD) was 4.5 × 103 colony-forming units (CFU)/mL for Salmonella species; 4.5 × 104 CFU/mL to differentiate Salmonella Enteritidis and Salmonella Typhimurium; and 2.3 × 103 CFU/mL for E. coli O157:H7, in a duplex assay. With a 6 h pre-enrichment, the LOD was 4.5 CFU/mL for Salmonella Enteritidis and Salmonella Typhimurium, and 2.3 CFU/mL for E. coli O157:H7. The cPCR amplification took only 14 min, and the NALF assay took ca. 5 min. The total analysis time was less than 20 min. Based on these observations, we propose that the developed assay is simple, ultra-fast, and applicable for on-site detection of foodborne pathogens.