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Diagnóstico viral desplegable en el campo utilizando CRISPR-Cas13
Cameron Myhrvold1,2, Catherine A Freije1,2,3, Jonathan S Gootenberg4,5,6,7,8
1Broad Institute of the Massachusetts Institute of Technology (MIT) and Harvard, Cambridge, MA 02142, USA. pardis@broadinstitute.org cmyhrvol@broadinstitute.org cfreije@broadinstitute.org.
Science (New York, N.Y.)
|April 28, 2018
Resumen
Una nueva plataforma de diagnóstico, SHERLOCK (desbloqueo de reportero enzimático específico de alta sensibilidad), detecta rápidamente el virus del Zika (ZIKV) y el virus del dengue (DENV) en muestras de pacientes. El protocolo HUDSON (calentamiento de muestras de diagnóstico no extraídas para eliminar nucleasas) permite la detección sin instrumentos en menos de dos horas.
Área de la Ciencia:
- Biología molecular
- Virología
- Biotecnología
Sus antecedentes:
- La mitigación global de enfermedades infecciosas requiere herramientas de diagnóstico sensibles, específicas y desplegables en el campo.
- Los métodos de diagnóstico existentes pueden carecer de la velocidad y la accesibilidad requeridas para una respuesta rápida al brote.
Objetivo del estudio:
- Evaluar la plataforma SHERLOCK basada en Cas13 para la detección sensible y específica del virus del Zika (ZIKV) y del virus del dengue (DENV).
- Desarrollar y validar el protocolo HUDSON para la detección viral rápida y sin instrumentos directamente a partir de muestras de pacientes.
- Evaluar la capacidad de SHERLOCK para diferenciar los serotipos DENV y las cepas ZIKV.
Principales métodos:
- Utilizó la plataforma SHERLOCK basada en Cas13 para la detección de ácido nucleico.
- Desarrolló e implementó el protocolo HUDSON para la preparación de muestras.
- Sensibilidad del ensayo a 1 copia por microlitro para ZIKV y DENV.
- Validación de la capacidad de SHERLOCK para distinguir los serotipos DENV y las cepas ZIKV.
Principales resultados:
- SHERLOCK detectó ZIKV y DENV en concentraciones tan bajas como 1 copia/μL.
- El protocolo HUDSON permitió la detección de DENV sin instrumentos en muestras de pacientes en menos de 2 horas.
- SHERLOCK diferenció con éxito los cuatro serotipos de DENV y las cepas pandémicas de ZIKV.
- Se logró un diseño rápido (< 1 semana) y pruebas de ensayos libres de instrumentos para polimorfismos virales de un solo nucleótido.
Conclusiones:
- La plataforma SHERLOCK, combinada con el protocolo HUDSON, ofrece una solución de diagnóstico sensible, específica y rápida para ZIKV y DENV.
- Este enfoque facilita la detección sin instrumentos en el punto de atención, crucial para la gestión de brotes de enfermedades infecciosas.
- La capacidad de la plataforma para detectar variantes virales específicas y polimorfismos de un solo nucleótido aumenta su utilidad en epidemiología molecular.
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