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.
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.
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