Search

Infectious diseases caused by Aeromonas salmonicida (A. salmonicida) have a huge impact and produce significant losses in aquaculture and fish farming. Fish pathogen early detection is a critical step for the rapid identification and prevention of these problems. This work presents a novel portable label-free ultrasensitive electrochemical immunosensor for A. salmonicida detection in seawater. It consists of a fluidic integrated electrochemical-cell-chip (ECC) with independent chambers enclosing three electrochemical cells (ECs). Anti-A. salmonicida (AbSalm) antibodies were covalently attached to the gold surface of the microfabricated electrodes and were used for the sensitive detection of A. salmonicida. The antibody-antigen immunoreaction was studied by enzyme-linked immunosorbent assay (ELISA), and the surface functionalization was characterized by using quartz crystal microbalance (QCM), differential pulse voltammetry (DPV), and electrochemical impedance spectroscopy (EIS). The performance of the developed immunosensor, in terms of sensitivity, repeatability, and specificity, was also studied. The linear working range varied between 1 and 10

Background: Ready-to-eat products, such as leafy greens, must be carefully controlled as they are directly consumed without any treatment to reduce the presence of potential pathogens. Food industries, especially those that process products with short shelf-life, demand rapid detection of foodborne pathogens such as Shiga Toxinproducing Escherichia coli (STEC). In this sense, molecular methods can fulfill both requirements of turnaround time and consumer safety. The most popular rapid methods are those based on real-time PCR (qPCR) however, vegetables contain inhibitory compounds that may inhibit the amplification reaction thus, there is a need for novel sample preparation protocols. Results: In the current study, a low-cost sample treatment based on sequential filtration steps was developed. This protocol was combined with covalent organic frameworks (COFs), and compared against a chelating resin, to evaluate their performance by multiplex qPCR targeting the major virulence genes of STEC, namely stx1, stx2, and eae, along with the rfbE for the specific identification of serogroup O157 due to its particularly high incidence, and an Internal Amplification Control to assess reaction inhibition. The optimized sample treatment effectively removed vegetable qPCR inhibitory compounds, and it was possible to detect STEC in spiked ready-toeat salad samples in one working day, roughly 5 h, with an LOD50 of 8.7 CFU/25 g with high diagnostic sensitivity and specificity. The method was also assessed in samples with cold-stressed bacteria with good results, further demonstrating its applicability. Significance: It was demonstrated for the first time that COFs are suitable for DNA extraction and purification. In addition to this, due to the tunable nature of these materials, it is envisioned that future modifications in terms of pore size or combination with magnetic materials, will allow to further improve their performance. In addition to this, the rapid and low-cost sample treatment protocol developed demonstrated suitable for the rapid screening of STEC vegetable samples. published

Nucleic acid amplification-based techniques have gained acceptance by the scientific, and general, community as reference methodologies for many different applications. Since the development of the gold standard of these techniques, polymerase chain reaction (PCR), back in the 1980s many improvements have been made, and alternative techniques emerged reporting improvements over PCR. Among these, isothermal amplification approaches resulted of particular interest as could overcome the need of specialized equipment to accurately control temperature changes, but it was after year 2000 that these techniques have flourished in a huge number of novel alternatives with many different degrees of complexities and requirements. An added value is their possibility to be combined with many different naked-eye detection strategies, simplifying the resources needed, allowing to reduce cost, and serving as the basis for novel developments of lab-on-chip systems, and miniaturized devices, for point-of-care testing. In this review, we will go over different types of naked-eye detection strategies, combined with isothermal amplification. This will provide the readers up-to-date information for them to select the most appropriate strategies depending on the particular needs and resources for their experimental setup.

6 pages, 4 tables, 1 figure.-- Under a Creative Commons license, Listeria monocytogenes is a major foodborne pathogen which mainly infects susceptible individuals through the consumption of contaminated foods. To this end, ready-to-eat (RTE) food products are of particular concern as this microorganism is widely distributed, can survive, and even grow, under adverse conditions, and thus must be carefully controlled. In the present study, an interlaboratory ring trial was organized to evaluate an open formula qPCR-based method for the detection of L. monocytogenes. The molecular method was evaluated on a novel RTE seafood product, developed in the framework of a European project, the SEAFOODAGE (EAPA_758/2018). Six laboratories located in Spain and Portugal participated in the study, and the results obtained indicated that this new method presented high diagnostic sensitivity (100%) reaching a low limit of detection (, This work was financially supported by the Seafood Age project, which was co-financed by the Interreg Atlantic Area Program (EAPA_758/2018) though the European Development Fund (ERDF). Mrs. Sarah Azinheiro was financed by a Ph.D. grant from the Fundação para a Ciência e a Tecnologia (SFRH/BD/140396/2018). Dr. Alejandro Garrido-Maestu and Luís D. R. Melo acknowledge funding from the Fundação para a Ciência e Tecnologia through the Scientific Employment Stimulus Program (2021.02810. CEECIND and 2021.00221. CEECIND, respectively). This study was supported by the Fundação para a Ciência e a Tecnologia (FCT) under the scope of the strategic funding of UIDB/04469/2020 unit

Salmonella spp. and antimicrobial resistant microorganisms are two of the most important health issues worldwide. In the present study, strains naturally isolated from mussels harvested in Galicia (one of the main production areas in the world), were genetically characterized attending to the presence of virulence and antimicrobial resistance genes. Additionally, the antimicrobial profile was also determined phenotypically. Strains presenting several virulence genes were isolated but lacked all the antimicrobial resistance genes analyzed. The fact that some of these strains presented multidrug resistance, highlighted the possibility of bearing different genes than those analyzed, or resistance based on completely different mechanisms. The current study highlights the importance of constant surveillance in order to improve the safety of foods.

Foodborne pathogens are still a significant source of morbidity and mortality worldwide. In addition to this the current methodologies to track these microorganisms cannot cope with the current intensive production systems, thus novel methods are of outmost importance. DNA-based methods have already demonstrated suitable to address this issue, but most of them are targeted methods such as real-time PCR (qPCR), meaning that one will only find what is looking for, thus taking the risk of missing relevant pathogens in a given sample. To overcome this limitation we have developed an easy-to-implement methodology which enables the detection of several pathogens simultaneously by using long-read Next Generation Sequencing (NGS) with MinION. The method was named “semi-targeted” due to the combination of a non-targeted detection method, NGS, with the usage of selective media in order to partially eliminate non-pathogenic interfering bacteria. To this end, we included an enrichment step for the recovery of different pathogens, namely Salmonella Enteritidis and Typhimurium, Listeria monocytogenes and Escherichia coli O157:H7, after DNA extraction and library preparation, the samples were analyzed with MinION implementing the low-cost Flongle Flow Cells. The methodology was successfully evaluated in spiked milk samples with an excellent agreement with the results obtained by qPCR and culture-based methods. The method can provide accurate results after only 2 h of sequencing. Sample multiplexing, along with the lower cost of the Flongle Flow Cells and the reduced price of the MinION platform, make the assay cost-effective that is of importance for the food industry. Starting the method with a classical microbiological approach, the enrichment, the method is easy to implement in testing laboratories, it provides flexibility in terms of potential pathogens to be detected, and the positive results can be easily confirmed following culture-based, or other type, of confirmation procedures.

Accurate and sensitive identification of DNA mutations in tumor cells is critical to the diagnosis, prognosis and personalized therapy of cancer. Conventional polymerase chain reaction (PCR)-based methods are limited by the complicated amplification process. Herein, an amplification-free surface enhanced Raman spectroscopy (SERS) approach which directly detects point mutations in cancer cells has been proposed. A highly sensitive and uniform SERS substrate was fabricated using gold@silver core-shell nanorods, achieving an enhancement factor of 1.85 × 106. By combining the SERS-active nanosubstrate with molecular beacon probes, the limit of detection reached as low as 50 fM. To enable parallel analysis and automated operation, the SERS sensor was integrated into a microfluidic chip. This novel chip-based assay was able to differentiate between mutated and wild-type KRAS genes among a variety of other nucleic acids from cancer cells in 40 min. Owing to the simple operation and fast analysis, the SERS-based DNA assay chip could potentially provide insights into clinical cancer theranostics in an easy and inexpensive manner at the point of care.

This article contains data related to the research article entitled “Novel approach for accurate minute DNA quantification on microvolumetric solutions” (Carvalho et al., 2018). The combination of PicoGreen® with a microvolume fluorospectrometer is a popular DNA quantification method due to its high sensitivity and minimal consumption of sample, being commonly used to evaluate the performance of microfluidic devices designed for DNA purification. In this study, the authors present data related with the effect of DNA fragmentation level. The present data article includes the data used on the precision evaluation, in terms of repeatability, of the mathematical models developed to obtain the standards curve for salmon sperm DNA (low molecular weight). In addition, results related with the effect of some compounds on the DNA quantification accuracy using λDNA are presented.

Lactococcus garvieae, Yersinia ruckeri and Flavobacterium psychrophilum are three of the most important pathogens in worldwide rainbow trout aquaculture (Oncorrhynchus mykiss Walbaum). In this work, a multiplex quantitative PCR (qPCR) method for the simultaneous detection of the three pathogens was developed and tested in a commercial trout farm. Fifty-four spleen and brain samples from a trout farm were analyzed using both multiplex qPCR and classic microbiological methods. When comparing qPCR results and classic plate diagnostic techniques no negative deviations were observed, which resulted in a 100% relative sensitivity of the multiplex qPCR method developed. In addition, efficiency of the qPCR method ranged between 97.5% and 108.8%. The specificity of the combination of primers and probes was successfully tested in 57 target and non-target bacterial strains Hence, the multiplex qPCR method developed in the present study could be used as a reliable diagnostic tool for the detection of L. garvieae, Y. ruckeri and F. psychrophilum in rainbow trout farms, enabling faster diagnostics (only a few hours) and contributing to a quick response and management of bacterial disease outbreaks.

Miniaturization of DNA-based techniques can bring interesting advantages for food analysis, such as portability of complex analytical procedures. In the olive oil industry, miniaturization can be particularly interesting for authenticity and traceability applications, through in situ control of raw materials before production and/or the final products. However, variety identification is challenging, and implementation on miniaturized settings must be carefully evaluated, starting from the selected analytical approach. In this work, SSR- and SNP-based genotyping strategies were investigated for the identification and differentiation of two olive varieties from the Northwest of Spain. For the selected SNPs two genotyping methods were tested: real-time allele-specific PCR and high resolution melting analysis. These methods were compared and evaluated regarding their potential for integration in a microfluidic device. Both SNP-based methods proved to be successful for identification of the selected varieties, however real-time allele-specific PCR was the one that achieved the best results when analyzing mixtures, allowing the identification of both monovarietal samples and mixtures of the varieties tested with up to 25%.

Lignin particles (LPs) have gained prominence due to their biodegradability and bioactive properties. LP production at nano and micro scale produced from organosolv lignin and the understanding of size's effect on their properties is unexplored. This work aimed to produce and characterize lignin nanoparticles and microparticles using a green synthesis process, based on ethanol-solubilized lignin and water. Spherical shape LPs, with a mean size of 75 nm and 215 nm and with a low polydispersity were produced, as confirmed by transmission electron microscopy and dynamic light scattering. LPs thermal stability improved over raw lignin, and the chemical structure of lignin was not affected by the production method. The antimicrobial tests proved that LPs presented a bacteriostatic effect on Escherichiacoli and Salmonella enterica. Regarding the antioxidant potential, LPs had a good antioxidant activity that increased with the reaction time and LPs concentration. LPs also presented an antioxidant effect against intracellular ROS, reducing the intracellular ROS levels significantly. Furthermore, the LPs showed a low cytotoxic effect in Caco-2 cell line. These results showed that LPs at different scales (nano and micro) present biological properties and are safe to be used in different high value industrial sectors, such as biomedical, pharmaceutical and food., This study was supported by the Portuguese Foundation for Science and Technology (FCT) under the scope of the strategic funding of UID/BIO/04469/2020 unit, BioTecNorte operation (NORTE-01-0145-FEDER000004) funded by the European Regional Development Fund under the scope of Norte2020 - Programa Operacional Regional do Norte, and MICRODIGEST project (grant agreement 037716) co-funded by FCT and ERDF through COMPETE2020., info:eu-repo/semantics/publishedVersion

Listeria monocytogenes continues to be a major challenge for the food industry due to its ubiquity and difficulty to be eliminated from processing facilities. The DNA amplification-based methods can overcome limitations of culture-based methods; however, due to the stability of the DNA, false positive results may occur, associated to the presence of harmless, dead microorganisms. The incorrect assessment of the results will have a huge impact on the producers in terms of stopping the production/distribution, leading to economic losses. In the current study, a multiplex RT-qPCR method was developed. The detection of mRNA allows for the specific detection of live bacteria. Additionally, two genetic targets (hly and actA) were co-amplified for improved specificity, along with an Internal Amplification Control to rule out false negative results due to reaction inhibition. The optimized methodology was evaluated in smoked salmon samples inoculated with different combinations and concentrations of live and dead bacteria. The method demonstrated high sensitivity (LOD50/LOD95 of 1.2/5.1 cfu/25 g). The performance of the method was compared against the reference standard ISO 11290, for which a Cohen's k of 0.94 was obtained, being interpreted as “almost complete concordance” among both methods. In addition, other parameters evaluated included the relative sensitivity, specificity, accuracy, as well as the positive and negative predictive values, all being above 90%. This next-day methodology can significantly reduce the time of analysis of culture-based methods and can specifically detect live L. monocytogenes.

Escherichia coli O157 continues to be the most prevalent serotype among the Shiga toxin-producing E. coli infection cases confirmed in Europe. The reference methodology to detect this pathogen is lengthy and time consuming, thus we sought to develop a novel method that has low instrumentation requirement, and allowed naked-eye detection. Isothermal amplification of bacterial DNA was performed by Recombinase Polymerase Amplification, and the addition of SYBR Green I (RPA-SG), which allowed the visualization of results with naked-eye under a UV lamp. The results obtained in spiked ground meat samples by RPA-SG compared favorably to qPCR (relative sensitivity, specificity and accuracy higher than 90%, and Cohen's k of 0.81), with a limit of detection of 19 cfu/25 g. The novel methodology outperformed a culture-based approach, where none of the typical colonies were confirmed as O157 due to high concentration of interfering microorganisms. These results were obtained in one working day (same-day detection), having an average time to completion of about 5 h, including enrichment, DNA extraction, amplification and detection.

Purification and concentration of DNA is a critical step on DNA-based analysis, which should ensure efficient DNA isolation and effective removal of contaminants that may interfere with downstream DNA amplification. Complexity of samples, minute content of target analyte, or high DNA fragmentation greatly entangles the success of this step. To overcome this issue, we designed and fabricated a novel miniaturized disposable device for a highly efficient DNA purification. The microfluidic device showed binding efficiency and elution yield of 90.1% and 86.7%, respectively. Moreover, the effect of DNA fragmentation, a parameter that has not been previously addressed, showed a great impact in the recovery step. The microfluidic system integrated micropillars with chitosan being used as the solid-phase for a pH-dependent DNA capture and release. We have showed the potential of the device in the successful purification of environmental DNA (eDNA) from river water samples contaminated with Dreissena polymorpha, an invasive alien species responsible for unquestionable economic and environmental consequences in river water basins. Additionally, the device was also able to concentrate the DNA extract from highly diluted samples, showing promising results for the early detection of such invasive species, which may allow prompt measures for a more efficient control in affected areas. Suitability for integration with downstream DNA analysis was also demonstrated through qPCR analysis of the samples purified with the microfluidic device, allowing detection of the target species even if highly diluted.

The continuous contamination of foods with L. monocytogenes, highlights the need for additional controls in the food industry. The verification of food processing plants is key to avoid cross-contaminations, and to assure the safety of the food products. In this study, a new methodology for the detection of L. monocytogenes on food contact surfaces was developed and evaluated. It combines Recombinase Polymerase Amplification (RPA) with the lateral flow (LF) naked-eye detection. Different approaches for the recovery of the bacteria from the surface, the enrichment step and downstream analysis by RPA-LF were tested and optimized. The results were compared with a standard culture-based technique and qPCR analysis. Sampling procedure with sponges was more efficient for the recovery of the bacteria than a regular swab. A 24 h enrichment in ONE broth was needed for the most sensitive detection of the pathogen. By RPA-LF, it was possible to detect 1.1 pg/&micro, L of pure L. monocytogenes DNA, and the complete methodology reached a LoD50 of 4.2 CFU/cm2 and LoD95 of 18.2 CFU/cm2. These results are comparable with the culture-based methodology and qPCR. The developed approach allows for a next-day detection without complex equipment and a naked-eye visualization of the results.

L. monocytogenes continues to be a major health issue in Europe, as well as worldwide. Faster methods, not only for detection, but also for sample preparation are of great interest particularly for this slow-growing pathogen. Immunomagnetic separation has been previously reported to be an effective way to concentrate bacteria, and remove inhibitors. In the present study, different commercial antibodies were evaluated to select the most appropriate one, in order to develop a highly specific method. Additionally, magnetic nanoparticles, instead of microparticles, were selected due to their reported advantages (higher surface-volume ration and faster kinetics). Finally, the separation protocol, with a calculated capture efficiency of 95%, was combined with real-time PCR for highly sensitive detection of the concentrated bacteria. The optimized IMS-qPCR allowed to reduce hands-on time in the sample treatment, without affecting the overall performance of the method as a very low limit of detection was still obtained (9.7 CFU/ 25 g) with values for sensitivity, specificity, accuracy, positive and negative predictive values of 100%, resulting in a kappa index of concordance of 1.00. These results were obtained in spiked food samples of different types (chicken, fish, milk, hard and fresh cheese), further demonstrating the applicability of the optimized methodology presented.

This work aims at the development and characterisation of bio-based active bi-layer films by solvent casting and electrospinning in order to be used for setting up active packaging solutions. Ethyl cellulose was used as the main material for the production of a layer on microfibrillated cellulose (MFC) films. This layer was formed with and without the incorporation of cinnamaldehyde (CNMA) which was used as antimicrobial compound. The MFC structures were obtained with a combination of different fibre dimensions and degree of fibrillation resulting in 16 different structures with porosities varying from 33 to 63%. A computational 3D simulation study of the porous structures was performed providing information about thickness, porosity and pore size uniformity and based on that a Picea abies-based MFC structure was selected. Structure characterization was evaluated using scanning electron microscopy, and pore dimensions were quantified using an image analysis tool. The bi-layer films chemical properties were studied using Fourier transform infrared spectroscopy and X-ray diffraction. Regarding barrier properties the bi-layer films produced by solvent casting were the ones showing a better barrier capacity. Both solvent casting and electrospinning processing showed to be useful to obtain a more hydrophobic surface (evaluated through contact angle measurements), being the higher values obtained for bi-layer films produced by electrospinning. Regarding colour parameters, the bi-layer films showed to be highly influenced by the production method and by the incorporation of CNMA. Regarding the antimicrobial activity, the bi-layer films with the incorporation of CNMA showed high antimicrobial activity against Listeria monocytogenes and Salmonella Typhimurium when the solvent casting method was used. Overall results showed that MFC-based films can be functionalised through the casting or electrospinning of ethyl cellulose solutions, aiming an antimicrobial and hydrophobic bi-layer film based on cellulose.

The optimization and evaluation of the performance of microfluidic devices for DNA purification requires the use of a reliable DNA quantification method. The samples collected from these devices usually have small volumes and, in the case of food, forensic and environmental applications, these samples are also complex, frequently containing highly fragmented DNA and minute concentrations. Therefore, combining a PicoGreen® assay with a microvolume fluorospectrometer is a popular technique for DNA quantification, providing highly sensitive quantification with minimal consumption of sample. However, this method has limitations, such as being affected by the degree of fragmentation of DNA and by the presence of some compounds commonly used in DNA extraction and purification protocols. In this work, these limitations and their influence on the accuracy of the quantification method were evaluated. Low molecular weight salmon sperm DNA was selected, being less purified and more fragmented than the λDNA standard most frequently used. It was shown that the standard curves generated with λDNA were not suitable for the quantification of fragmented DNA, such as DNA from highly processed samples and/or samples that have been exposed to harsh environments. In addition, a mathematical model was developed to find a better adjustment for the standard curve, for the salmon sperm DNA samples. This approach can be used as a tool to overcome important limitations found in this quantification method, allowing to include more data in the standard curve or test different mathematical models to better fit the standards data.

Listeriosis continues to be a major health issue. This is demonstrated by the fact that, even though efforts have been made, its incidence does not decrease. Furthermore, in Europe, over 2014 a 30% increase was reported respect to 2013. In the present study two isothermal DNA amplification methods, one based on Loop-mediated isothermal AMPlification (qLAMP), and the other on Recombinase Polymerase Amplification (RPA), were developed and extensively evaluated. Both techniques demonstrated their reliability to detect Listeria monocytogenes in different types of foods. The method included a two-step enrichment, which additionally reduces the chances of detecting dead bacteria. Over the evaluation with pure bacterial DNA, the qLAMP and RPA methods resulted 10 to 100 times less sensitive than qPCR (with two different detection chemistries), but when tested in real food samples the results showed very good concordance with those obtained by qPCR and by selective agar plating (index kappa of concordance between 0.90 and 0.95). Additionally, a very low limit of detection (below 10 CFU/25 g) was obtained. Thus the optimal performance of both isothermal techniques, and their adequacy for their implementation in the food industry, was demonstrated.

The safety of using nano- and microparticles is a developing concern. In this study, we conducted risk assessments of chitosan microparticles (CMs) using in vitro human epithelial cell lines and in vivo animal model, Caenorhabditis elegans. After engineering of various CMs, we screened four CMs based on antimicrobial activity, which is a potential usage for disease treatment caused by multidrug resistant bacteria, and evaluated for risk assessments. CMs, with strong antimicrobial activity, and inorganic nanoparticles (SiO2, TiO2, and ZnO) did not cause toxicity in human cells measured by cell membrane integrity, mitochondria activity, and reactive oxygen species concentration. However, when applied to C. elegans, only CMs generated with low molecular weight chitosan and tripolyphosphate at 0.1% did not affect the lifespan, while the other CMs and inorganic nanoparticles shortened the lifespan, suggesting that they may cause subtle toxicity. These results suggest that C. elegans could be a sensitive animal model to measure low level of toxicity of nano- and microparticles. Taken together, although CMs do not cause toxicity at working concentrations of antimicrobial activity in human epithelial cells, they may cause toxicity at high concentration, suggesting that nano- and microparicles should be thoroughly investigated before they are applied.

In the present study a multiplex real-time PCR method was developed for early detection of diseased fish infected by Aeromonas salmonicida, Vibrio anguillarum, and/or Tenacibaculum maritimum. The method consisted of the detection of three species-specific genes after DNA extraction with a commercial kit. Three types of samples were tested, and the results were compared with those of traditional diagnosis. The method obtained a limit of detection of 104 cfu/mL (2 x 102 cfu/tube). Additionally, 27 samples from fish showing signs of disease were correctly diagnosed by the developed methodology, demonstrating its suitability for implementation in aquaculture.

Campylobacteriosis is one of the most important reported zoonosis worldwide. Besides poultry other sources of infection have been described. In the current study, the incidence of Campylobacter spp. was assessed over a five-month period in mussel samples harvested from one of the most important producing areas (Galicia, NW Spain) in Europe. Out of 91 samples screened, 8% were positive and identified as C. lari by MALDI-TOF and whole genome sequencing. All were detected during the colder months (February and March). The antimicrobial resistance and virulence genes analysis indicated that all were multi-resistant to at least 4 antimicrobials. They were negative for the presence of 5 virulence-related genes. This is the first report of this zoonotic pathogen in mussels from one of the most important shellfish producing regions in Europe. The genomes of these 7 C. lari isolates were released to the genome public database at NCBI.

Nowadays, the most widely accepted rapid methods in food microbiology rely on nucleic acid amplification such as PCR/real-time PCR. A major claimed limitation of these methods is their incapacity to differentiate among viable and non-viable microorganisms. In the present study we report the development of a novel multiplex loop-mediated isothermal amplification method which, by combining immunomagnetic separation, to concentrate and purify the bacteria, along with propidium monoazide to block the amplification of DNA from non-viable microorganisms. The method allowed to specifically detect viable Listeria monocytogenes present in milk products. We designed an internal amplification control to rule out false negative results due to reaction inhibition, which is differentiated from L. monocytogenes by a simple melt curve analysis. Overall, the methodology provided results higher than 95% in terms of sensitivity, specificity and accuracy, as well as a Cohen's k of 0.97, reaching a limit of detection of 2.7 cfu/25 g. In samples inoculated with up to 106-107 cfu of dead microorganisms, the method demonstrated capable of effectively eliminating undesired amplification.

SARS-CoV-2 is the coronavirus responsible for COVID-19, which has spread worldwide, affecting more than 200 countries, infecting over 140 million people in one year. The gold standard to identify infected people is RT-qPCR, which is highly sensitive, but needs specialized equipment and trained personnel. The demand for these reagents has caused shortages in certain countries. Isothermal nucleic acid techniques, such as loop-mediated isothermal amplification (LAMP) have emerged as an alternative or as a complement to RT-qPCR. In this study, we developed and evaluated a multi-target RT-LAMP for the detection of SARS-CoV-2. The method was evaluated against an RT-qPCR in 152 clinical nasopharyngeal swab samples. The results obtained indicated that both assays presented a “good concordance” (Cohen’s k of 0.69), the RT-LAMP was highly specific (99%) but had lower sensitivity compared to the gold standard (63.3%). The calculated low sensitivity was associated with samples with very low viral load (RT-qPCR Cq values higher than 35) which may be associated with non-infectious individuals. If an internal Cq threshold below 35 was set, the sensitivity and Cohen’s k increased to 90.9% and 0.92, respectively. The interpretation of the Cohen’s k for this was “very good concordance”. The RT-LAMP is an attractive approach for frequent individual testing in decentralized setups.