A Sensitive and Specific Fluorescent RT-LAMP Assay for SARS-CoV-2 Detection in Clinical Samples.

The raging COVID-19 pandemic has created an unprecedented demand for frequent and widespread testing to limit viral transmission. Reverse transcription loop-mediated isothermal amplification (RT-LAMP) has emerged as a promising diagnostic platform for rapid detection of SARS-CoV-2, in part because it can be performed with simple instrumentation. However, isothermal amplification methods frequently yield spurious amplicons even in the absence of a template. Consequently, RT-LAMP assays can produce false positive results when they are based on generic intercalating dyes or pH-sensitive indicators. Here, we report the development of a sensitive RT-LAMP assay that leverages on a novel sequence-specific probe to guard against spurious amplicons. We show that our optimized fluorescent assay, termed LANTERN, takes only 30 min to complete and can be applied directly on swab or saliva samples. Furthermore, utilizing clinical RNA samples from 52 patients with COVID-19 infection and 21 healthy individuals, we demonstrate that our diagnostic test exhibits a specificity and positive predictive value of 95% with a sensitivity of 8 copies per reaction. Hence, our new probe-based RT-LAMP assay can serve as an inexpensive method for point-of-need diagnosis of COVID-19 and other infectious diseases.

An engineered CRISPR-Cas12a variant and DNA-RNA hybrid guides enable robust and rapid COVID-19 testing.

Extensive testing is essential to break the transmission of SARS-CoV-2, which causes the ongoing COVID-19 pandemic. Here, we present a CRISPR-based diagnostic assay that is robust to viral genome mutations and temperature, produces results fast, can be applied directly on nasopharyngeal (NP) specimens without RNA purification, and incorporates a human internal control within the same reaction. Specifically, we show that the use of an engineered AsCas12a enzyme enables detection of wildtype and mutated SARS-CoV-2 and allows us to perform the detection step with loop-mediated isothermal amplification (LAMP) at 60-65 °C. We also find that the use of hybrid DNA-RNA guides increases the rate of reaction, enabling our test to be completed within 30 minutes. Utilizing clinical samples from 72 patients with COVID-19 infection and 57 healthy individuals, we demonstrate that our test exhibits a specificity and positive predictive value of 100% with a sensitivity of 50 and 1000 copies per reaction (or 2 and 40 copies per microliter) for purified RNA samples and unpurified NP specimens respectively.