loop-mediated isothermal amplification (lamP) as a point-of-care SarS-covdetection method

Pravovremeno testiranje većeg broja ljudi na SARS-CoV-2 virus je povezano sa nižim mortalitetom od COVID-19 oboljenja. Međutim, većina zemalja nema mogućnosti za takvo masivno testiranje putem metode “PCR u realnom vremenu”, zbog visoke cene neophodne opreme i potrebe za stručnim osobljem. Zbog toga se razvijaju brze i ekonomičnije metode, koje se najčešće zasnivaju na izotermalnim metodama amplifikacije DNK. Za ove metode nije potreban ciklični termostat, zbog čega su primenljivije za terensku upotrebu. Fokus je na izotermalnoj amplifikaciji posredovanoj petljom (lamP), zbog njene visoke specifičnosti, mogućnosti za upotrebu neprečišćenih uzoraka i jednostavnosti merenja signala. Autori predstavljaju pregled najvažnijih radova o LAMP metodi za detekciju SARS-CoV-2 virusa objavljenih tokom 2020. godine, kao i opise nekoliko komercijalnih kompleta na bazi LAMP metode za COVID-19 testiranje.Massive testing for SARS-CoV-2 virus is related to lower mortality rates of COVID-19. Most countries face challenges to perform massive testing with the currently available methods (real-time PCR), due to expensive equipment and requirement of highly skilled personnel. To overcome these challenges, faster and cost-effective alternative detection methods are being developed, primarily based on isothermal methods of nucleic acid amplification (isoNAAs). PCR depends on precision instruments, high cleanliness of operating conditions and cannot be easily used on-site, while isoNAAs, which do not require thermal cyclers, are more applicable for point-of-care (PoC) use. Loop-mediated isothermal amplification (LAMP) is one of the isoNAA most in focus for COVID-19 tests due to its high specificity and possibility to use unpurified specimens in combination with simplified detection setup. The article gives a review of the most significant publications on use of LAMP for SARS-CoV-2 detection and of the several commercial LAMP-based COVID-19 testing kits.Ministarstva prosvete, nauke i tehnološkog razvoja Republike Srbije – broj ugovora 451-03-68/2020-14/ 200358 za autore M. Dj, T.K i Lj.J i broj ugovora 451-03-9/2021- 14/200125 za Ž.D.P. Autori zahvaljuju Jeleni Ognjenov na pomoći u pripremi ilustracija za rad

Cultivating Multidisciplinarity: Manufacturing and Sensing Challenges in Cultured Meat Production

Meat cultivation via cellular agriculture holds great promise as a method for future food production. In theory, it is an ideal way of meat production, humane to the animals and sustainable for the environment, while keeping the same taste and nutritional values as traditional meat and having additional benefits such as controlled fat content and absence of antibiotics and hormones used in the traditional meat industry. However, in practice, there is still a number of challenges, such as those associated with the upscale of cultured meat (CM). CM food safety monitoring is a necessary factor when envisioning both the regulatory compliance and consumer acceptance. To achieve this, a multidisciplinary approach is necessary. This includes extensive development of the sensitive and specific analytical devices i.e., sensors to enable reliable food safety monitoring throughout the whole future food supply chain. In addition, advanced monitoring options can help in the further optimization of the meat cultivation which may reduce the currently still high costs of production. This review presents an overview of the sensor monitoring options for the most relevant parameters of importance for meat cultivation. Examples of the various types of sensors that can potentially be used in CM production are provided and the options for their integration into bioreactors, as well as suggestions on further improvements and more advanced integration approaches. In favor of the multidisciplinary approach, we also include an overview of the bioreactor types, scaffolding options as well as imaging techniques relevant for CM research. Furthermore, we briefly present the current status of the CM research and related regulation, societal aspects and challenges to its upscaling and commercialization

Using Vertebrate Stem and Progenitor Cells for Cellular Agriculture, State-of-the-Art, Challenges, and Future Perspectives

Global food systems are under significant pressure to provide enough food, particularly protein-rich foods whose demand is on the rise in times of crisis and inflation, as presently existing due to post-COVID-19 pandemic effects and ongoing conflict in Ukraine and resulting in looming food insecurity, according to FAO. Cultivated meat (CM) and cultivated seafood (CS) are protein-rich alternatives for traditional meat and fish that are obtained via cellular agriculture (CA) i.e., tissue engineering for food applications. Stem and progenitor cells are the building blocks and starting point for any CA bioprocess. This review presents CA-relevant vertebrate cell types and procedures needed for their myogenic and adipogenic differentiation since muscle and fat tissue are the primary target tissues for CM/CS production. The review also describes existing challenges, such as a need for immortalized cell lines, or physical and biochemical parameters needed for enhanced meat/fat culture efficiency and ways to address them

Molecular Approaches for Detection of <i>Trichoderma</i> Green Mold Disease in Edible Mushroom Production

Due to the evident aggressive nature of green mold and the consequently huge economic damage it causes for producers of edible mushrooms, there is an urgent need for prevention and infection control measures, which should be based on the early detection of various Trichoderma spp. as green mold causative agents. The most promising current diagnostic tools are based on molecular methods, although additional optimization for real-time, in-field detection is still required. In the first part of this review, we briefly discuss cultivation-based methods and continue with the secondary metabolite-based methods. Furthermore, we present an overview of the commonly used molecular methods for Trichoderma species/strain detection. Additionally, we also comment on the potential of genomic approaches for green mold detection. In the last part, we discuss fast screening molecular methods for the early detection of Trichoderma infestation with the potential for in-field, point-of-need (PON) application, focusing on isothermal amplification methods. Finally, current challenges and future perspectives in Trichoderma diagnostics are summarized in the conclusions

Bioengineering Outlook on Cultivated Meat Production

Cultured meat (also referred to as cultivated meat or cell-based meat)—CM—is fabricated through the process of cellular agriculture (CA), which entails application of bioengineering, i.e., tissue engineering (TE) principles to the production of food. The main TE principles include usage of cells, grown in a controlled environment provided by bioreactors and cultivation media supplemented with growth factors and other needed nutrients and signaling molecules, and seeded onto the immobilization elements—microcarriers and scaffolds that provide the adhesion surfaces necessary for anchor-dependent cells and offer 3D organization for multiple cell types. Theoretically, many solutions from regenerative medicine and biomedical engineering can be applied in CM-TE, i.e., CA. However, in practice, there are a number of specificities regarding fabrication of a CM product that needs to fulfill not only the majority of functional criteria of muscle and fat TE, but also has to possess the sensory and nutritional qualities of a traditional food component, i.e., the meat it aims to replace. This is the reason that bioengineering aimed at CM production needs to be regarded as a specific scientific discipline of a multidisciplinary nature, integrating principles from biomedical engineering as well as from food manufacturing, design and development, i.e., food engineering. An important requirement is also the need to use as little as possible of animal-derived components in the whole CM bioprocess. In this review, we aim to present the current knowledge on different bioengineering aspects, pertinent to different current scientific disciplines but all relevant for CM engineering, relevant for muscle TE, including different cell sources, bioreactor types, media requirements, bioprocess monitoring and kinetics and their modifications for use in CA, all in view of their potential for efficient CM bioprocess scale-up. We believe such a review will offer a good overview of different bioengineering strategies for CM production and will be useful to a range of interested stakeholders, from students just entering the CA field to experienced researchers looking for the latest innovations in the field

Loop-mediated isothermal amplification (LAMP) assay coupled with gold nanoparticles for colorimetric detection of Trichoderma spp. in Agaricus bisporus cultivation substrates

Abstract One of the significant challenges in organic cultivation of edible mushrooms is the control of invasive Trichoderma species that can hinder the mushroom production and lead to economic losses. Here, we present a novel loop-mediated isothermal amplification (LAMP) assay coupled with gold nanoparticles (AuNPs) for rapid colorimetric detection of Trichoderma spp. The specificity of LAMP primers designed on the tef1 gene was validated in silico and through gel-electrophoresis on Trichoderma harzianum and non-target soil-borne fungal and bacterial strains. LAMP amplification of genomic DNA templates was performed at 65 °C for only 30 min. The results were rapidly visualized in a microplate format within less than 5 min. The assay is based on salt-induced aggregation of AuNPs that is being prevented by the amplicons produced in case of positive LAMP reaction. As the solution color changes from red to violet upon nanoparticle aggregation can be observed with the naked eye, the developed LAMP-AuNPs assay can be easily operated to provide a simple initial screening for the rapid detection of Trichoderma in button mushroom cultivation substrate

Mammalian Cell-Growth Monitoring Based on an Impedimetric Sensor and Image Processing within a Microfluidic Platform

In recent years, advancements in microfluidic and sensor technologies have led to the development of new methods for monitoring cell growth both in macro- and micro-systems. In this paper, a microfluidic (MF) platform with a microbioreactor and integrated impedimetric sensor is proposed for cell growth monitoring during the cell cultivation process in a scaled-down simulator. The impedimetric sensor with an interdigitated electrode (IDE) design was realized with inkjet printing and integrated into the custom-made MF platform, i.e., the scaled-down simulator. The proposed method, which was integrated into a simple and rapid fabrication MF system, presents an excellent candidate for the scaled-down analyses of cell growths that can be of use in, e.g., optimization of the cultivated meat bioprocess. When applied to MRC-5 cells as a model of adherent mammalian cells, the proposed sensor was able to precisely detect all phases of cell growth (the lag, exponential, stationary, and dying phases) during a 96-h cultivation period with limited available nutrients. By combining the impedimetric approach with image processing, the platform enables the real-time monitoring of biomasses and advanced control of cell growth progress in microbioreactors and scaled-down simulator systems