Simple, Inexpensive RNA Isolation and One‐Step RT‐qPCR Methods for SARS‐CoV‐2 Detection and General Use

The most common method for RNA detection involves reverse transcription followed by quantitative polymerase chain reaction (RT‐qPCR) analysis. Commercial one‐step master mixes—which include both a reverse transcriptase and a thermostable polymerase and thus allow performing both the RT and qPCR steps consecutively in a sealed well—are key reagents for SARS‐CoV‐2 diagnostic testing; yet, these are typically expensive and have been affected by supply shortages in periods of high demand. As an alternative, we describe here how to express and purify Taq polymerase and M‐MLV reverse transcriptase and assemble a homemade one‐step RT‐qPCR master mix. This mix can be easily assembled from scratch in any laboratory equipped for protein purification. We also describe two simple alternative methods to prepare clinical swab samples for SARS‐CoV‐2 RNA detection by RT‐qPCR: heat‐inactivation for direct addition, and concentration of RNA by isopropanol precipitation. Finally, we describe how to perform RT‐qPCR using the homemade master mix, how to prepare in vitro−transcribed RNA standards, and how to use a fluorescence imager for endpoint detection of RT‐PCR amplification in the absence of a qPCR machine In addition to being useful for diagnostics, these versatile protocols may be adapted for nucleic acid quantification in basic research. © 2021 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Preparation of a one‐step RT‐qPCR master mix using homemade enzymes Basic Protocol 2: Preparation of swab samples for direct RT‐PCR Alternate Protocol 1: Concentration of RNA from swab samples by isopropanol precipitation Basic Protocol 3: One‐step RT‐qPCR of RNA samples using a real‐time thermocycler Support Protocol: Preparation of RNA concentration standards by in vitro transcription Alternate Protocol 2: One‐step RT‐PCR using endpoint fluorescence detectio

Revista de educación

Monográfico con el título: 'La formación práctica de estudiantes universitarios : repensando el Prácticum'. Resumen basado en el de la publicaciónSe describe una experiencia de Innovación Educativa dirigida a mejorar la formación práctica del futuro profesional de la Educación Social, realizada por un grupo responsable de la tutoría de la Universidad y alumnado de la Universidad del País Vasco. El objetivo principal se centra en la revisión e innovación del Prácticum actual en la Escuela Universitaria de Magisterio de Bilbao, donde la Diplomatura de Educación Social se imparte desde 1998, atendiendo promociones de 150 alumnos. Respondiendo a las necesidades emanadas en el marco del Espacio Europeo de Educación Superior, el plan se perfila conforme a una estructura modular e interdisciplinar. Seguidamente se describe la metodología cualitativa desarrollada, donde se conjugan el modelo de investigación-acción colaborativo y el pensamiento reflexivo mediante la técnica del relato. Se utilizan otras técnicas y estrategias de recogida de información, entre los que destacan grupos de discusión, escritura automática, grabaciones en audio. En tercer lugar, se exponen los resultados más relevantes en dos ámbitos: el referido a la metodología, y el referido a la innovación. Se define una competencia específica referida al saber ser estar, con objeto de lograr una identidad más precisa del profesional de la Educación Social. Finalmente, y a la luz de las necesidades detectadas, se plantean distintas propuestas de mejora para dar continuidad al trabajo desarrollado, y que se recogen en el plan de acción, basado en las competencias definidas y en la subsiguiente concreción de tareas.MadridBiblioteca de Educación del Ministerio de Educación, Cultura y Deporte; Calle San Agustín, 5 - 3 Planta; 28014 Madrid; Tel. +34917748000; [email protected]

CRISPR Systems for COVID-19 Diagnosis

The emergence of the new coronavirus 2019 (COVID-19) was first seen in December 2019, which has spread rapidly and become a global pandemic. The number of cases of COVID-19 and its associated mortality have raised serious concerns worldwide. Early diagnosis of viral infection undoubtedly allows rapid intervention, disease management, and substantial control of the rapid spread of the disease. Currently, the standard approach for COVID-19 diagnosis globally is the RTqPCR test; however, the limited access to kits and associated reagents, the need for specialized lab equipment, and the need for highly skilled personnel has led to a detection slowdown. Recently, the development of clustered regularly interspaced short palindromic repeats (CRISPR)-based diagnostic systems has reshaped molecular diagnosis. The benefits of the CRISPR system such as speed, precision, specificity, strength, efficiency, and versatility have inspired researchers to develop CRISPRbased diagnostic and therapeutic methods. With the global COVID-19 outbreak, different groups have begun to design and develop diagnostic and therapeutic programs based on the efficient CRISPR system. CRISPR-based COVID-19 diagnostic systems have advantages such as a high detection speed (i.e., 30 min from raw sample to reach a result), high sensitivity and precision, portability, and no need for specialized laboratory equipment. Here, we review contemporary studies on the detection of COVID-19 based on the CRISPR system

Streamlined inactivation, amplification, and Cas13-based detection of SARS-CoV-2

The COVID-19 pandemic has highlighted that new diagnostic technologies are essential for controlling disease transmission. Here, we develop SHINE (Streamlined Highlighting of Infections to Navigate Epidemics), a sensitive and specific diagnostic tool that can detect SARS-CoV-2 RNA from unextracted samples. We identify the optimal conditions to allow RPA-based amplification and Cas13-based detection to occur in a single step, simplifying assay preparation and reducing run-time. We improve HUDSON to rapidly inactivate viruses in nasopharyngeal swabs and saliva in 10 min. SHINE’s results can be visualized with an in-tube fluorescent readout — reducing contamination risk as amplification reaction tubes remain sealed — and interpreted by a companion smartphone application. We validate SHINE on 50 nasopharyngeal patient samples, demonstrating 90% sensitivity and 100% specificity compared to RT-qPCR with a sample-to-answer time of 50 min. SHINE has the potential to be used outside of hospitals and clinical laboratories, greatly enhancing diagnostic capabilities