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FnCas9 Editor Linked Uniform Detection Assay (FELUDA)
Science behind the Test:
The assay uses a combination of CRISPR Cas9 biology and paper strip chemistry to produce a visible read-out of COVID-19 infection status. Briefly, samples are collected from the suspected individual in virus transport medium (VTM). Using routine RNA isolation methods, viral RNA sample will contain viral RNA is extracted from the sample. Following this, the isolated RNA is converted into complementary DNA (cDNA) and amplified by Polymerase Chain Reaction (PCR) in a single step using special chemistry where all viral RNA molecules will be labelled with a chemical group called biotin. If the sample is negative, no amplification of viral DNA will occur in this step. Following PCR, the amplified products are mixed with an indigenously characterised (CSIR IGIB) CRISPR CAS9 protein-RNA complex that has been genetically and chemically modified. Design of the CRISPR complex has been done in such a way that it will make complex only with COVID DNA but will not recognise non-COVID DNA. This property makes it highly specific and accurate in diagnosing COVID 19 infection and differentiating it from other infections such as influenza. When it mixed with amplified products. This complex solution is then applied to a special paper strip that uses gold nanoparticles to carry the CRISPR complex and associated viral DNA. Both COVID amplified material and chemically tagged CRISPR complex will move together upwards on the paper strip. Chemistry of a small part of the paper strip has been designed in such a way that during the flow COVID amplified products will get deposited in that portion. Now if there is complex in the flowing solution, the deposited area will develop a colour that can be visually seen. If there is some other non COVID DNA such a complex will not form and no color will be seen. The duration of color development after the DNA is deposited on the strip is between 2-5 min. In addition to this band (called test band) there is an additional band that develops on the strip called control band which comes up in every strip indicating successful assay.
This assay thus uses a cutting edge CRISPR tool, combines high tech science for a low tech application that can cater to people in remote areas where complex instrumentation is difficult to arrange. This assay is thus a rapid implementation of an extremely powerful gene editing technology and has applications in a large number of areas beyond COVID-19 diagnosis.
Advantages of the Test
1) Accuracy of the test as good as accuracy with gold standard qRT-PCR.
Only nucleic acid tests provide accurate results on current infection status. Current test is based on nucleic acid detection of the Virus.
2) Test run time is less than 1 hour, makes it as faster as antigen rapid test.
Rapid antigen tests have low sensitivity but high specificity and misses out samples with low viral load. Antibody tests provide post infection status in individuals.
3) Test result interpretation is based on colour development on paper strip, thus can be interpreted by minimum qualified technicians.
This CRISPR based assay eliminates the need for expensive quantitative real time machine and provides direct visual readout on a paper strip.
4) Test needs only PCR machine thus test can be performed in low resourced areas
Cost of a PCR machine is less than 1 lakh and manufactured in India in large quantities. Moreover, Indigenous battery driven PCR machines are available in the country.
5) It uses high end science, but can cater to low resource settings with less expenditure.
6) Simple design and nature of ordinary ingredients of the test allow for mass scale production in a short time.
Intended Use of the Test.
The intended use of the product will be for detection of COVID-19 positive cases from RNA samples of individuals. A single test from RNA takes less than 1h to complete and has a sensitivity/specificity of 96% and 98% respectively. |