Browsing by Author "Matute, Tamara "

Now showing 1 - 5 of 5
  • Thumbnail Image
    Item
    Accessible LAMP-Enabled Rapid Test (ALERT) for Detecting SARS-CoV-2
    (2021) Bektaş, Ali ; Covington, Michael F. ; Aidelberg, Guy ; Arce, Anibal ; Matute, Tamara ; Núñez, Isaac; Walsh, Julia ; Boutboul, David ; Delaugerre, Constance ; Lindner, Ariel B. ; Federici, Fernán ; Jayaprakash, Anitha D.
    The coronavirus disease 2019 (COVID-19) pandemic has highlighted bottlenecks in large-scale, frequent testing of populations for infections. Polymerase chain reaction (PCR)-based diagnostic tests are expensive, reliant on centralized labs, can take days to deliver results, and are prone to backlogs and supply shortages. Antigen tests that bind and detect the surface proteins of a virus are rapid and scalable but suffer from high false negative rates. To address this problem, an inexpensive, simple, and robust 60-minute do-it-yourself (DIY) workflow to detect viral RNA from nasal swabs or saliva with high sensitivity (0.1 to 2 viral particles/mu L) and specificity (>97% true negative rate) utilizing reverse transcription loop-mediated isothermal amplification (RT-LAMP) was developed. ALERT (Accessible LAMP-Enabled Rapid Test) incorporates the following features: (1) increased shelf-life and ambient temperature storage, compared to liquid reaction mixes, by using wax layers to isolate enzymes from other reagents; (2) improved specificity compared to other LAMP end-point reporting methods, by using sequence-specific QUASR (quenching of unincorporated amplification signal reporters); (3) increased sensitivity, compared to methods without purification through use of a magnetic wand to enable pipette-free concentration of sample RNA and cell debris removal; (4) quality control with a nasopharyngeal-specific mRNA target; and (5) co-detection of other respiratory viruses, such as influenza B, by multiplexing QUASR-modified RT-LAMP primer sets. The flexible nature of the ALERT workflow allows easy, at-home and point-of-care testing for individuals and higher-throughput processing for labs and hospitals. With minimal effort, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-specific primer sets can be swapped out for other targets to repurpose ALERT to detect other viruses, microorganisms, or nucleic acid-based markers.
  • No Thumbnail Available
    Item
    Building an Open-Source DNA Assembler Device
    (2023) Orostica, Boris; Nunez, Isaac; Matute, Tamara; Nunez, Felipe; Federici, Fernan
    This article introduces an open-source thermal cycling machine designed specifically for Golden Gate DNA assembly. The prototype device can achieve efficiency similar to a commercial PCR thermocycler.
  • No Thumbnail Available
    Item
    Decentralizing Cell-Free RNA Sensing With the Use of Low-Cost Cell Extracts
    (2021) Arce, Anibal; Guzman Chavez, Fernando; Gandini, Chiara; Puig, Juan; Matute, Tamara; Haseloff, Jim; Dalchau, Neil; Molloy, Jenny; Pardee, Keith; Federici, Fernan
    Cell-free gene expression systems have emerged as a promising platform for field-deployed biosensing and diagnostics. When combined with programmable toehold switch-based RNA sensors, these systems can be used to detect arbitrary RNAs and freeze-dried for room temperature transport to the point-of-need. These sensors, however, have been mainly implemented using reconstituted PURE cell-free protein expression systems that are difficult to source in the Global South due to their high commercial cost and cold-chain shipping requirements. Based on preliminary demonstrations of toehold sensors working on lysates, we describe the fast prototyping of RNA toehold switch-based sensors that can be produced locally and reduce the cost of sensors by two orders of magnitude. We demonstrate that these in-house cell lysates provide sensor performance comparable to commercial PURE cell-free systems. We further optimize these lysates with a CRISPRi strategy to enhance the stability of linear DNAs by knocking-down genes responsible for linear DNA degradation. This enables the direct use of PCR products for fast screening of new designs. As a proof-of-concept, we develop novel toehold sensors for the plant pathogen Potato Virus Y (PVY), which dramatically reduces the yield of this important staple crop. The local implementation of low-cost cell-free toehold sensors could enable biosensing capacity at the regional level and lead to more decentralized models for global surveillance of infectious disease.
  • Thumbnail Image
    Item
    Open educational resources for distributed hands-on teaching in molecular biology
    (2025) Cerda Rojas, Ariel Patricio; Castillo-Navarrete, Juan Luis; Aravena Lazo, Alejandro Daniel; Zapata Romero, Valentina Paz; Arce Medina, Anibal Andres; Araya, Wladimir ; Gallardo, Domingo; Aviles, Javiera; Quero, Francisco; Nuñez, Isaac; Matute, Tamara ; Navarro, Felipe ; Blanco Zepeda, Marta Victoria; Velozo Caballero, Sebastian Ignacio; Rodriguez, Sebastian; Aguilera, Sebastian; Chateau Gannon, Francisco; Olivares Donoso, Ruby Carolina; Ramirez-Sarmiento, Cesar; Federici, Fernan
    The urgent need to develop a more equitable bioeconomy has positioned biotechnology capacity building at the forefront of international priorities. However, in many educational institutions, particularly in low- and middle-income countries, this remains a major challenge due to limited access to reagents, equipment, and technical documentation. In this work, we describe Open Educational Resources (OER) composed of locally produced biological reagents, open source hardware and free software to teach fundamental techniques in biotechnology such as LAMP DNA amplification, RT-PCR RNA detection, enzyme kinetics and fluorescence imaging. The use of locally produced reagents and devices reduces costs by up to one order of magnitude. During the pandemic lockdowns, these tools were distributed nationwide to students’ homes as a lab-in-a-box for remote teaching of molecular biology. To test their performance, a total of 93 undergraduate students tested these resources during a biochemistry practical course. 27 out of 31 groups (~87%) successfully achieved the objectives of the PCR activity, while 28 out of 31 groups (~90%) correctly identified the target using LAMP reactions. To assess the potential application in secondary school, we organized three workshops for high school teachers from different institutions across Chile and performed an anonymous questionnaire, obtaining a strong agreement on how these OER broaden teachers’ perspectives on the techniques and facilitate the teaching of molecular biology topics. This effort was made possible through a close collaboration with open source technology advocates and members of DIYbio communities, whose work is paving the way for low-cost training in biology. All the protocols and design files are available under open source licenses
  • Thumbnail Image
    Item
    Recombinant protein expression and purification of codon-optimizedBst-LF polymerase v1
    (2020) Rivera, Maira ; Cazaux, Severine; Cerda, Ariel ; Arce Medina, Anibal ; Núñez, Isaac ; Matute, Tamara ; Brown, Alex ; Gasulla, Javier ; Federici, Fernan ; Ramirez-Sarmiento, Cesar A.