Browsing by Author "Torres, Fernando"

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    A novel viral RNA detection method based on the combined use of trans-acting ribozymes and HCR-FRET analyses
    (2024) da Silva, Leonardo Ferreira; Garay, Aisel Valle; Queiroz, Pedro Felipe; de Resende, Sophia Garcia; Gomide, Mayna; de Oliveira, Izadora Cristina Moreira; Bernasol, Amanda Souza; Arce, Anibal; Santos, Liem Canet; Torres, Fernando; Silva-Pereira, Ildinete; de Freitas, Sonia Maria; Coelho, Cintia Marques
    The diagnoses of retroviruses are essential for controlling the rapid spread of pandemics. However, the real-time Reverse Transcriptase quantitative Polymerase Chain Reaction (RT-qPCR), which has been the gold standard for identifying viruses such as SARS-CoV-2 in the early stages of infection, is associated with high costs and logistical challenges. To innovate in viral RNA detection a novel molecular approach for detecting SARS-CoV-2 viral RNA, as a proof of concept, was developed. This method combines specific viral gene analysis, trans-acting ribozymes, and Fluorescence Resonance Energy Transfer (FRET)-based hybridization of fluorescent DNA hairpins. In this molecular mechanism, SARS-CoV-2 RNA is specifically recognized and cleaved by ribozymes, releasing an initiator fragment that triggers a hybridization chain reaction (HCR) with DNA hairpins containing fluorophores, leading to a FRET process. A consensus SARS-CoV-2 RNA target sequence was identified, and specific ribozymes were designed and transcribed in vitro to cleave the viral RNA into fragments. DNA hairpins labeled with Cy3/Cy5 fluorophores were then designed and synthesized for HCR-FRET assays targeting the RNA fragment sequences resulting from ribozyme cleavage. The results demonstrated that two of the three designed ribozymes effectively cleaved the target RNA within 10 minutes. Additionally, DNA hairpins labeled with Cy3/Cy5 pairs efficiently detected target RNA specifically and triggered detectable HCR-FRET reactions. This method is versatile and can be adapted for use with other viruses. Furthermore, the design and construction of a DIY photo-fluorometer prototype enabled us to explore the development of a simple and cost-effective point-of-care detection method based on digital image analysis.
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    Citizen Science and Phytosanitary Surveillance Systems Are Complementary Tools to Follow the Invasion of Harmonia axyridis
    (2023) Grez, Audrey A.; Zaviezo Palacios, Tania; Orellana, Ignacio; Pino, Luna; Roy, Helen E.; Torres, Fernando; Rebolledo, Claudia
    Citizen science is a valuable tool for early detection, distribution, and spread of invasive alien species (IAS). Nevertheless, citizen science initiatives have several potential biases and may be complemented with long-term structured monitoring schemes. We analyzed the spatial-temporal dynamics of the invasion of Harmonia axyridis (Pallas) (Coleoptera: Coccinellidae) in Chile, based upon two citizen sciences databases (WEB and INAT) and one structured monitoring (SAG). We collected 8638 H. axyridis occurrences between 2009 and 2020. WEB had a higher number of records than SAG and INAT, and in all databases, the number of records has increased over time. The three databases showed that the invasion started in central Chile and then spread toward the north and south. The WEB and SAG recorded occurrences in the extreme north and south, whereas INAT concentrated all the occurrences in a more limited area, included in WEB and SAG. Both citizen science initiatives concentrated their records in areas of high human populations whereas SAG records had a more even distribution across regions. At 2020, WEB accounted for 55%, SAG 54%, and INAT 8% of the total area accumulated with H. axyridis, with only 16% of area shared among databases. WEB and INAT obtained most of their records in urban and industrial land cover types, while SAG records were more evenly represented in different land cover types. Our results confirm that combined methods, including citizen science initiatives, national surveillance system, and localized samplings, complement each other in providing knowledge to understand the patterns, processes, and consequences of this invasion.