10/​05/​2020

Feluda Test – A new rapid test method for the identification of SARS-CoV-2

Just recently, the TATA Group received approval for the commercial launch of “Feluda” (acronym for FNCas9 Editor Limited Uniform Detection Assay) by the Drugs Controller Gerneral of India.

What is Feluda?

The Feluda test is the first CRISPR/​Cas9 (Clustered Regularly Inter­spaced Short Palin­dromic Repeats) rapid test available for COVID-​​19 detection. With this new qPCR independent point-​​of-​​care diagnostic approach, the genomic sequence of SARS-​​CoV-​​2 can be detected by Milenias lateral flow assay HybriDetect.

The paper strip-​​based test was developed at the Council of Scien­tific and Indus­trial Research’s constituent lab, the Institute of Genomics and Integrative Biology (CSIR-​​IGIB) based in New Delhi, India. The testing kit addresses the urgent need for accurate COVID-​​19 mass testing and has many advan­tages compared to the gold standard, qPCR. Like SHERLOCK (specific high-​​sensitivity enzymatic reporter unlocking) which is a COVID 19 test using Cas12 for detection of SARS-​​CoV-​​2, developed by scien­tists from the Broad Institute in the US, the name Feluda refers to an Indian fictive detective (film director: Satyajit Ray).

Advan­tages of the Feluda test compared to qPCR:

  • Afford­ability
  • Can be used in settings with limited resources
  • Less time to result (around 45 minutes)
  • No qPCR equipment (no need for expensive devices)
  • High ease of use
  • Lateral Flow based readout for easy inter­pre­tation of results

 

With 96% sensi­tivity and 98% speci­ficity, Feluda’s analytical perfor­mance is compa­rable to the qPCR results.

How the Feluda test worksThe COVID-​​19 test developed in New Delhi, India by a research team led by Debojyoti Chakraborty and Souvik Maiti is based on a Cas9 enzymatic readout for detecting nucleotide sequences and identi­fying nucle­obase identity. Just a few weeks ago, Emmanuelle Charp­entier and Jennifer Doudna have been awarded the 2020 Nobel Prize in Chemistry for their devel­opment of CRISPR/​Cas9 gene editing technology. The Cas9 readout used for coron­avirus detection has requirement of trans-​​cleavage activity of reporter molecules like Cas12 or Cas13 methods.

The scien­tists from CSIR-​​IGIB used Cas9 from Francisella novicida (FNCas9), which shows very high mismatch sensi­tivity and can distin­guish between nucleotide sequences differing by only one mismatch. The principle of the paper strip test was origi­nally designed for the identi­fi­cation of sickle cell anemia, a disease caused by a point mutation, and adapted to COVID-​​19 testing due to the Coron­avirus outbreak resulting in an urgent need for mass testing.

The FnCas9 used in this new method does not produce collateral activity on substrates due to the use of a catalyt­i­cally inactive FnCas9-​​gRNA-​​complex.

Therefore, it is an affinity based method and no trans-​​cleavage signal output is generated.

Fig. 1: Feluda test principle on HybriDetect. FnCas9 with FAM labeled gRNA binds to biotiny­lated sample if result is positive.

The Feluda test and Milenia HybriDetect

The indian researchers designed a gRNA, labeled with FAM, which is important for the visual­ization of the test line on Milenia HybriDetect (Figure 2). After RNA extraction, the first step of the paper strip test is an optimized single step Reverse Transctription-​​PCR (RT-​​PCR), or alter­na­tively a RPA protocol, where the sample gets amplifi­cated and biotiny­lated. In the next step, the FnCas9 gRNA (labeled with FAM) is incubated with the biotiny­lated substate (virus sequence if sample is positive). Due to the strep­ta­vidin, immobi­lized on the HybriDetect dipstick, the RNP Complex (CRISPR ribonu­cle­o­protein) binds to the test line (Figure 2).

The RNP-​​complex bound to the labeled substrate is visualized with anti-​​Fam antibodies conju­gated to gold nanopar­ticles and a positive test line is formed if substrate is bound to the RNP-​​complex. Summa­rized this means, two lines for a positive and a single line for a negative test result.

The visual inter­pre­tation of the HybriDetect dipstick is as easy as the inter­pre­tation of a pregnancy test and therefore no trained personnel is required.

Fig.2: Milenia HybriDetect and FnCas9. The test is positive if the biotiny­lated DNA is present and binds to strep­ta­vidin on the T-​​Line. FnCas9 binds to the biotiny­lated DNA and is captured by FAM antibodies coated with gold nanopar­ticles for visual­ization.

When to expect Feluda Test to be available?

The Union Health Minister of India Harsh Vardhan said on October 11th, that the rollout of the new Covid-​​19 test is expected in the next few weeks. The test developed by scien­tists from the Council of Scien­tific and Indus­trial Research’s Institute of Genomics and Integrative Biology in New Delhi is going to provide results in 45 minutes and is priced around Rs 500 ( ~ 5,70 Euro).

Update: the Feluda Test is already in use in India.

Additional Infor­mation on the Feluda test

  • Compared to other CRISPR methods for COVID Detection, including SHERLOCK and DETECTR, Feluda does not need a reporter and is therefore less complex.
  • The LOD of indias new test method is 10 copies of purified viral sequence as the authors mention in their latest publi­cation
  • To assist the detection of COVID-​​19 the scien­tists developed a smart­phone app named TOPSE (True Outcome Predicted via Strip Evalu­ation) which gives a predictive score based on background correction. Due to the stoichio­metric binding affinity of FnCas9 RNP to the target, a semi-​​quantitative readout of Feluda is possible with TOPSE.

Feluda for at home testing

The Scien­tists from CSIR-​​IGIB are currently working on a Feluda version for at home coron­avirus testing. Therefore they are trying to do a machine free, RPA based ampli­fi­cation of the virus genome. The aim is an end-​​to end instru­men­tation free testing protocol.

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