Tamal Das, TIFR Hyderabad
To this day, quantitative reverse transcription-polymerase chain reaction (qRT-PCR) remains the most widespread method for detecting SARS-Cov-2 coronavirus, whose genetic information is carried by ribonucleic acids (RNAs). In qRT-PCR, viral RNAs become templates for a specific enzyme called reverse transcriptase (RT), which generates the corresponding DNA sequences. Using this DNA as starting material, another enzyme called DNA polymerase then kick-starts a cyclic chain reaction called polymerase chain reaction or PCR. PCR eventually increases the DNA concentration by more than a billion-fold, making it detectable by a fluorescent dye. In spite of being the gold standard for the Covid-19 test, qRT- PCR has certain disadvantages. It needs pricey instruments, it needs trained man-power, and it takes hours to get the test done. With millions of people getting infected with the virus, we need a bed-side test kit, and for that, we need an instrument-independent method that is faster, cheaper, and preferably more sensitive than qRT-PCR.
One such method is RT-LAMP. In RT-LAMP, scientists do use reverse transcriptase (RT) to generate DNAs out of viral RNAs, but they replace the subsequent PCR-based DNA amplification by a different loop-mediated amplification (LAMP) reaction. While PCR requires precise up-and-down ramping of temperature in each cycle of amplification, LAMP reaction can happen under constant temperature around 70ºC. This feature liberates the test from the stringency of PCR machines and allows the reaction to happen over a simple hot plate. Another simplicity of RT-LAMP comes from its detection strategy. For the detection of DNA amplification, the reaction mixture contains a pH-sensitive dye that changes its color from red to yellow upon successful amplification. Thus, in RT-LAMP test, a yellow solution implies a COVID-positive sample while a red solution implies a negative one. Further, to make the process even simpler, researchers from Harvard Medical School have recently invented an
inexpensive pipeline that eliminates the rigorous RNA isolation step. With this invention, it looks like RT-LAMP should be able to tell us whether a person is Covid-19 positive or not within 30 minutes.
Other than RT-LAMP, there is, of course, another method that promises to transcend the disadvantages of qRT-PCR. This method reengineers the CRISPR-based gene editing technology for viral diagnostic and is called Specific High Sensitivity Enzymatic Reporter UnLOCKing or SHERLOCK – an acronym inspired by the famous fictional detective character. SHERLOCK does not generate DNAs, but here viral RNAs get directly amplified and then, get detected by an enzyme called Cas13. This entire RNA amplification-detection process can take place in a commercially-available paper dipstick, making it perfect for bedside testing. To make it even faster, researchers at MIT have recently come up with a step SHERLOCK test, called STOPCovid. Interestingly, we also have an indigenous version of SHERLOCK. A team of scientists based on CSIR-IGIB in Delhi is now proposing a rapid FNCAS9 Editor Linked Uniform Detection Assay or ‘FELUDA’, after the fictional detective character of Satyajit Ray. In the latest development, Tata Sons have agreed to help in commercializing the FELUDA paper test kit and make it available for public use by June this year.
- Rabe and Cepko, SARS-CoV-2 Detection Using an Isothermal Amplification Reaction and a Rapid, Inexpensive Protocol for Sample Inactivation and Purification(link is external), medRxiv (Apr 2020)
- SARS-CoV-2/COVID-19 Detection Methods Based on CRISPR/Cas(link is external)
- Joung et al., Point-of-care testing for COVID-19 using SHERLOCK diagnostics(link is external)
- Azhar et al., Rapid, field-deployable nucleobase detection and identification using FnCas9(link is external), bioRxiv (Apr 2020)
[Last update 14 May 2020]