FnCas9 Editor Linked Uniform Detection Assay (FELUDA) for COVID-19:
This test assay is developed by CSIR Institute of Genomics and Integrative Biology, New Delhi with Tata sons.
The assay uses a combination of CRISPR Cas9 biology and paper strip chemistry to produce a visible read-out of COVID-19 positive cases.
(Read more if you like technical terms)
Briefly, RNA from patient is converted into complementary DNA (cDNA) and amplified by Polymerase Chain Reaction (PCR) in a single step. The primers used in this process contain a biotin group resulting in biotin-labelled amplicons at the end of the PCR step. The primers are designed targeting specific regions of the SARS-CoV2 sequence such that there is amplification only in COVID-19 positive cases. If the sample is negative, no amplification occurs in this step. Following PCR, the amplified products are incubated with catalytically inactive (also called dead) FnCas9 protein:dual guide RNA Ribonucleoprotein (RNP) complex. The dual guide RNA (gRNA) contains a Fluorescein amidite (FAM) group attached to it. The dFnCas9 has a very high specificity for its target which is determined by the sequence of the gRNA which is designed to recognize specific regions (20 nucleotides long) in the CoV2 sequence that was amplified in the PCR step (Acharya, Mishra, Paul et al. 2019, PNAS). Once the FAM labelled RNP recognizes its target, it forms a complex with the biotin labelled substrate (COVID-19 positive case). If it doesn’t find its target it does not form a complex with biotin labelled substrate (COVID-19 negative case). This complex is then applied to a paper strip which contains gold nanoparticles conjugated with anti-FAM antibody. These gold nanoparticles recognize the FAM label on the RNP, attach to them and move up the strip by lateral flow (aided by buffer in which the strip is dipped). As the complex moves up, it first encounters a region where Streptavidin molecules are immobilized. If the complex has biotin (due to biotinylated DNA from a COVID-19 positive case), it gets bound to this streptavidin region and produces a visually detectable signal (test line).
Remaining molecules migrate up to a control line which contains an antibody against the anti-FAM antibody producing a visually detectable control signal (control line). Due to the absence of biotin labelled primer products in COVID-19 negative sample, no test line is produced. The control line appears in both the cases working as a quality control for the paper-strip.
Test performs with similar specificity/sensitivity/accuracy as gold stand qRT-PCR test (>95%).