Fast molecular methods for the detection of spoilage fungi (in food products)

In the present thesis, the detection of spoilage and mycotoxigenic fungi in different food matrices was achieved in 24-48 h, providing a faster alternative to the conventional culture-based techniques which require up to 7 days from sampling to result. Consequently, the developed methods present an interesting alternative for the food industry resulting in reduction of costs associated with lengthy analyses and product recalls. Small modifications of the proposed methodology including enrichment time and temperature as well as sample size have proven to contribute to higher sensitivity and/or specificity which gives the developed methodology the required flexibility to fit the needs of the food industry. Furthermore, the different methods showcased different advantages. In particular, the real-time PCR assays have proven to be more sensitive; however, they require a real-time thermocycler in order to be performed. On the other hand, the isothermal amplification techniques coupled with naked-eye detection could be used for POC testing and early screening since they only require a heating device; providing in many cases a comparable sensitivity to the qPCR assays

Interlaboratory validation of a multiplex qPCR method for the detection of listeria monocytogenes in a ready-to-eat seafood product

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 (<10CFU/25 g) with an overall agreement with the reference method, attending to the Cohen's k, of 0.97 that is interpreted as almost complete agreement.info:eu-repo/semantics/publishedVersio

Multifuntional Gold Nanoparticles for the SERS Detection of Pathogens Combined with a LAMP–in–Microdroplets Approach

We developed a droplet-based optofluidic system for the detection of foodborne pathogens. Specifically, the loop-mediated isothermal amplification (LAMP) technique was combined with surface-enhanced Raman scattering (SERS), which offers an excellent method for DNA ultradetection. However, the direct SERS detection of DNA compromises the simplicity of data interpretation due to the variability of its SERS fingerprints. Therefore, we designed an indirect SERS detection method using multifunctional gold nanoparticles (AuNPs) based on the formation of pyrophosphate generated during the DNA amplification by LAMP. Towards this goal, we prepared multifunctional AuNPs involving three components with key roles: (1) thiolated poly(ethylene glycol) as stabilizing agent, (2) 1-naphthalenethiol as Raman reporter, and (3) glutathione as a bioinspired chelating agent of magnesium (II) ions. Thus, the variation in the SERS signal of 1-naphthalenethiol was controlled by the aggregation of AuNPs triggered by the complexation of pyrophosphate and glutathione with free magnesium ions. Using this strategy, we detected Listeria monocytogenes, not only in buffer, but also in a food matrix (i.e., ultra-high temperaturemilk) enabled by the massive production of hotspots as a result of the self-assemblies that enhanced the SERS signal. This allowed the development of a microdroplet-LAMP-SERS platform with isothermal amplification and real-time identification capabilities

Optimization and Clinical Evaluation of a Multi-Target Loop-Mediated Isothermal Amplification Assay for the Detection of SARS-CoV-2 in Nasopharyngeal Samples

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