Rapid, multiplex detection of SARS-CoV-2 using isothermal amplification coupled with CRISPR-Cas12a

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 (similar to 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 (similar to 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/mu L will enable the robust, fast detection of the virus in a dedicated equipment which is a major step towards population-wide accessible testing.