Many approaches have been adopted to mitigate SARS-CoV-2 infection and manage COVID-19’s spread. An essential component of pandemic control and future prevention is extensive testing at the point of care. This enables enhanced detection, making subsequent isolation measures and healthcare access for the infected individuals possible.
The qRT-PCR (quantitative reverse-transcriptase-polymerase chain reaction), employed for RNA detection in clinical samples, provides high sensitivity; the limit of detection is ~1 molecule/µl. However, this technique is too complex to implement for rapid point-of-care testing. In an alternative approach to the PCR-based methods, CRISPR-Cas proteins have been employed by a large team of researchers to identify the SARS-CoV-2 genomic RNA in patient samples with PCR-derived cycle threshold (Ct) values up to 22 – corresponding to ~1600 copies/µl in the assay.
This study was recently posted on the medRxiv* preprint server. Here, the researchers showed that unrelated CRISPR nucleases could be deployed in tandem to provide both direct RNA sensing and rapid signal generation, thus enabling robust detection of ∼30 RNA copies/microliter in 20 minutes. Thus, the assay – Fast Integrated Nuclease Detection In Tandem (FIND-IT) – combines RNA-guided Cas13 and Csm6 with a chemically stabilized activator and creates a one-step RNA detection for effective use at point-of-care.
On a mobile phone-based imaging device, the researchers showed that this assay detects as low as 200 cp/µl of target RNA within 30 minutes. However, they noted that increased speed and sensitivity of the one-pot detection chemistries are still needed to enable their widespread use for point-of-care diagnostics. In addition to sensitivity and speed, to meet the accuracy threshold set by the Food and Drug Administration (FDA) for emergency use authorization, the researchers analyzed this assay in replicates. They established that the assay met the 95% accuracy requirement for 20 replicates at 31 cp/µl of viral RNA in 60 min (the limit of detection as defined by the FDA).
The researchers also developed a detector consisting of a microfluidic chip with reaction chambers, a heating module that maintains the reactions at 37˚C, and a compact fluorescence imaging system. This enables ease of use at the point of care. They also demonstrated that the FIND-IT assay could detect SARS-CoV-2 genomic sequences in total RNA extracted from nasopharyngeal patient samples, with PCR-derived Ct values up to 29 in microfluidic chips, using a compact imaging system.
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