Assay for Sickle Cell Anemia Is Repurposed to Diagnose COVID-19
Assay for Sickle Cell Anemia Is Repurposed to Diagnose COVID-19

Assay for Sickle Cell Anemia Is Repurposed to Diagnose COVID-19

A CRISPR-based, paper-strip test developed by Indian researchers performs as well as real-time PCR in identifying the presence of SARS-CoV-2 and returns results within an hour.

G.B.S.N.P. Varma
Nov 18, 2020

ABOVE: Feluda-based detection of SARS-CoV-2
DR. DEBOJYOTI CHAKRABORTY’S LAB, CSIR-IGIB

In 1965, the famous Indian filmmaker Satyajit Ray created a beloved fictional detective named Feluda. In a series of short stories, the astute gumshoe tracks a vandal trying to destroy the ancient temples of India, busts a counterfeit medicine racket in Kathmandu, and cleans Bombay smugglers’ clocks. Inspired by the great analytical skills of the detective, researchers at the Delhi-based Council of Scientific and Industrial Research (CSIR)-Institute of Genomics and Integrative Biology have given their new CRISPR-based diagnostic for COVID-19 the affectionate nickname of Feluda (short for FnCas9 Editor Linked Uniform Detection Assay). TataMD Check, the commercial name of the test powered by Feluda, was launched on November 9 in India.

The test uses the CRISPR-Cas system to probe for genetic sequences specific to the SARS-CoV-2 virus in samples from nasal swabs or saliva applied to a paper strip. Like the fictional Feluda, it is fast, accurate, and works in all manner of field settings, according to a preprint posted to medRxiv in September. The regulatory agencies in India conducted an independent evaluation of the assay on patient samples and found Feluda to be highly accurate in detecting COVID-19 infections.

“The combination of precision, speed, and affordability in this technology is remarkable,” says Prashant Yadav, a healthcare supply-chain expert and senior fellow at the Center for Global Development in Washington, DC, a think tank largely supported by the Bill & Melinda Gates Foundation.

The Drugs Controller General of India approved Feluda for commercial use on September 19, and in an October 22 advisory, the Indian Council of Medical Research approved its use by public and private laboratories. The test costs about 500 rupees ($6.75 US) to make and process in the lab for a patient sample and gives results within an hour.

“Once validated at large scale and commercial manufacturing issues are dealt with, it can become the mainstay of COVID-19 testing, not only in India but many other countries in the world who are seeking a fast, affordable, and precise test,” says Yadav, who is not involved in the project.

India’s first reported case of COVID-19 was on January 30, and the first death on March 12.In the beginning of the pandemic, testing was limited to a few publicly funded laboratories in India. Later on, it was extended to private labs. Still, capacity is limited. India currently tests only around 0.1 percent of its population of 1.4 billion each day, causing the country to rank among the lowest in the world on a per-capita basis.

The US has nearly as many tests per day—around 1.2 million, compared with India’s 1.5 million—but a population that is smaller by a factor of four, explains Gautam Menon, a professor of physics and biology at Ashoka University and at the Institute of Mathematical Sciences in India who was not involved in the research. “I would think that we should test about 0.5 percent of our population every day, as an ideal, but we are testing more than ever before,” he says.

Earlier this year, a team of researchers at the Pune-based biotech company MyLab Discovery Solutions developed a test kit—PathoDetect—for COVID-19 that returns results in 2.5 hours with 100 percent accuracy. It was the first real-time PCR test for the novel coronavirus made in India that was approved in the country. It costs about $16. For PathoDetect, a significant limiting factor is the cost of the real-time PCR machine itself, which can be around $26,000 US.

Feluda, however, uses a paper strip to reveal results, similar to the readout of a pregnancy test. Anyone who can run a normal PCR machine and follow a simple pipetting protocol can do this, says co-lead scientist Debojyoti Chakraborty. Feluda can be performed at any diagnostic lab or healthcare center that houses a simple PCR machine and its equipment and reagents are less expensive than PathoDetect’s are.

The assay has been tested on more than 2,000 samples. It detected those who have SARS-CoV-2, a measure of sensitivity, 96 percent of the time, while ruling out those who didn’t have the virus, a measure of its specificity, 98 percent of the time.

From sickle cell anemia to COVID-19

Feluda emerged out of “serendipity,” says Chakraborty. For the past two-and-a-half years, he and his colleague Souvik Maiti had been working on a Cas9 system that could detect the mutation in the gene that causes sickle cell anemia for point-of-care CRISPR diagnostics. To test their approach, they traveled to far-flung healthcare centers in Chattisgarh state in India and collected saliva samples from people known to have sickle cell anemia as well as from potential silent carriers of the mutation.

As they were readying the publication of their results in January, COVID-19 hit, and they wondered if they could repurpose the technology to detect SARS-CoV-2.

Starting right away, Chakraborty says, his team of graduate students and postdocs worked day and night, with some colleagues falling sick with the virus along the way. By March, they had developed a prototype diagnostic, and in May, they licensed the technology to TATA Medical and Diagnostics Limited, which ushered it through regulatory approval.

Feluda works by first converting viral RNA from a sample to DNA, and then amplifying specific viral genes via PCR, during which it puts a biotin tag on the amplified products. Following this, the sample is incubated in the same PCR tube with a fluorescently tagged FnCas9–guide RNA complex, which looks for these sequences from SARS-CoV-2 and hooks onto them, creating a larger complex of the fluorescent FnCas9 and biotin-labeled viral DNA sequence. Then, the technician adds a buffer and dips a paper strip into the tube. Gold nanoparticles on the paper recognize the fluorescent tag and the whole complex flows up the strip and gets deposited on a line of streptavidin, which interacts with the biotin label on the DNA. The accumulation of the nanoparticles produces the visible signal.

There are a handful of other CRISPR-based tests for COVID-19, including one developed by MIT and Harvard University, and these employ Cas12 or Cas13 proteins to find the virus.

Harikrishnan Jayamohan, a research scientist at the University of Utah who was not involved in the work, says, “If Feluda works on a large scale in India, this would be a good alternative to RT-PCR. This would also be a big jump over antigen tests, which are widely-used, cheap, fast, and easy to use but are of very low sensitivity.” Nearly 50 percent of India’s COVID-19 diagnostics are done through antigen testing.

“Feluda is good in certain ways,” says Rakesh Kumar Mishra, the director of Hyderabad-based CSIR-Centre for Cellular and Molecular Biology who was not involved in the Feluda research. It will be particularly useful in remote places because Feluda can work with a simple PCR machine and that machine can be housed in rural healthcare center. But the real value of a paper-strip test is when it can be used for self-testing or at home, Yadav says, although Feluda is not ready for that yet.

“This is a tough thing we’re fighting,” says Mishra. “Any tool is a good tool.”

M. Azhar et al., “Rapid, accurate, nucleobase detection using FnCas9,” medRxiv, doi:10.1101/2020.09.13.20193581, 2020.