Efficient detection of the virus that causes COVID-19 has been a question on the minds of many researchers worldwide since early 2020. NCBS researcher Dr. Arati Ramesh, along with Dr. Akash Gulyani from the University of Hyderabad, sought to tackle the pressing need for a rapid, accurate, and inexpensive testing method. A team of students from NCBS and DBT-inStem joined their efforts to create PHANTOM (PHAsed NASBA-Translation Optical Method). PHANTOM provides a direct and ultrasensitive test that is both a powerful and an easily accessible strategy to detect a COVID-19 infection.
PHANTOM efficiently detects the presence of viral RNA in human patient samples, with clear distinction from samples designated negative for the virus. This diagnostic tool was developed using RNA biosensors to detect SARS-CoV-2 RNA, based on “toehold switches”, sensors which can be designed to detect any sequence of interest. The target RNA, in this case, a portion of the SARS-CoV-2 RNA, binds to the sensor and activates it. This activation then triggers production of a reporter protein that causes a change in colour (lacZ) or luminescence (Nano-lantern). The colour change is visible to the human eye, and to a mobile phone camera as well. The luminescence can be quantified using a luminometer. “This ease of detection was one of the big motivations behind developing this assay. One can simply look at the tube and tell whether the sample is COVID positive or not,” says Dr. Arati Ramesh.
The early steps of designing the biosensor involved computational design of a panel of potential sensors that might bind portions of the SARS-CoV-2 genomic RNA. Next, the team started narrowing down the potential biosensors from 1000s to a single sensor. By coupling the sensor to a step of isothermal RNA amplification, the sensitivity of the assay was amplified so that it can detect the infection down to a 100 copies of SARS-CoV-2-RNA. The assay has been validated against standard RT-qPCR tests.The biosensor efficiently detects the presence of viral RNA in swab samples from human patients, and this response correlates well with the cycle threshold values from RT-qPCR testing. Apart from high sensitivity, there is no sign of false negatives, the sensor is highly specific as well. “The power of the test is that it is sensitive and specific but there is no need for sophisticated equipment or trained personnel to interpret the results unlike the RT-PCR test which is considered the gold standard for SARS-CoV-2 detection,” says Dr. Akash Gulyani.
The PHANTOM platform does not require any sophisticated equipment and is usable even in a low resource setting. The entire test takes a minimum of 2 and a half hours, by which time a clear colour change is observed in COVID positive samples. An additional advantage is the potential to quantify the viral load in the sample, by measuring the amount of colour or luminescence.
The long term projection of this work is that the pipeline set up by Dr. Arati Ramesh and Dr. Akash Gulyani’s labs, can be easily re-purposed for other diagnostics and RNA and DNA biomarker sensing in future.
Prof. Satyajit Mayor, Centre Director, NCBS says, “Given its use as a platform technology that can be used in a resource-poor environment, it has attracted great interest from a number of organisations at the very start of the pandemic: CSR funds from PNB Housing, IQVIA, and a generous grant from APPI have supported this effort that we deemed a priority. It was conceived at the inception of the COVID-19 pandemic. Today, barely a few months on, efforts to make this technology into a product are ongoing at a furious pace and we are hopeful that several companies will come forward to take this on.”