SARS-CoV-2 Direct Detection Without RNA Isolation With Loop-Mediated Isothermal Amplification (LAMP) and CRISPR-Cas12

Frontiers in Medicine

Bertha Rueda-Zarazua¹  Alfredo Garcia-Venzor¹  Jorge Melendez-Zajgla¹  Vilma Maldonado²  Irma Lopez³  Hiram Olivera³  Eduardo Marquez-Garcia⁴  Angelica Moncada-Morales⁴  Joaquin Zuñiga⁴ 

[1] Cancer Functional Genomics Laboratory, Instituto Nacional de Medicina Genómica, Mexico City, Mexico;Epigenetics Laboratory, Instituto Nacional de Medicina Genómica, Mexico City, Mexico;Instituto Nacional de Referencia Epidemiológica, Mexico City, Mexico;Molecular Biology Unit, Instituto Nacional de Enfermedades Respiratorias, Mexico City, Mexico;

关键词: COVID-19;    SARS-CoV-2;    diagnosis;    LAMP;    CRISPR-Cas12a;   

DOI  :  10.3389/fmed.2021.627679

来源: Frontiers

【 摘 要 】

As to date, more than 49 million confirmed cases of Coronavirus Disease 19 (COVID-19) have been reported worldwide. Current diagnostic protocols use qRT-PCR for viral RNA detection, which is expensive and requires sophisticated equipment, trained personnel and previous RNA extraction. For this reason, we need a faster, direct and more versatile detection method for better epidemiological management of the COVID-19 outbreak. In this work, we propose a direct method without RNA extraction, based on the Loop-mediated isothermal amplification (LAMP) and Clustered Regularly Interspaced Short Palindromic Repeats-CRISPR associated protein (CRISPR-Cas12) technique that allows the fast detection of SARS-CoV-2 from patient samples with high sensitivity and specificity. We obtained a limit of detection of 16 copies/μL with high specificity and at an affordable cost. The diagnostic test readout can be done with a real-time PCR thermocycler or with the naked eye in a blue-light transilluminator. Our method has been evaluated on a small set of clinical samples with promising results.

【 授权许可】

CC BY