Rapid, multiplex detection of SARS-CoV-2 using isothermal amplification coupled with CRISPR-Cas12a
Diogo Figueiredo, António Cascalheira, João Gonçalves
Abstract
In December 2019 an outbreak erupted due to the beta coronavirus Severe Acute Respiratory Syndrome Coronavirus 2 in Wuhan, China. The disease caused by this virus (COVID-19) rapidly spread to all parts of the globe leading to a global pandemic. Efforts to combat the pandemic rely on RT-qPCR diagnostic tests that have high turnaround times (~ 24 h), are easily contaminated, need specialized equipment, facilities, and personnel that end up increasing the overall costs of this method. Loop-mediated isothermal amplification (LAMP) coupled with a reverse transcription step (RT-LAMP) is an alternative diagnostic method that can easily overcome these obstacles, when coupled with CRISPR/Cas it can eliminate false positives. Here we report a fast (~ 40 min), highly sensitive, point-of-care multiplex RT-LAMP and CRISPR/Cas12a assay to detect SARS-CoV-2. This fluorescence-based test achieved 100% specificity and 93% sensitivity using 25 positives and 50 negative patient samples for Ct < 35. Our reported LoD of 3 copies/µL will enable the robust, fast detection of the virus in a dedicated equipment which is a major step towards population-wide accessible testing.