California [US], August 6 (ANI): A research team led by scientists at the University of California, Berkeley, is aiming to develop a diagnostic test that is much faster and easier to deploy than the RT-PCR test which is today's gold standard for detecting COVID-19.
The research aims to combine two different types of CRISPR enzymes to create an assay that can detect small amounts of viral RNA in less than an hour.
The findings of the research were published in the journal 'Nature Chemical Biology'.
The research team is being led by Jennifer Doudna, among others. Doudna notably shared the 2020 Nobel Prize in Chemistry for the invention of CRISPR-Cas9 genome editing.
Frequent, rapid testing for COVID-19 is critical to controlling the spread of outbreaks, especially as new, more transmissible variants emerge. While the RT-PCR test uses qRT-PCR -- quantitative reverse-transcriptase-polymerase chain reaction (PCR) -- is extremely sensitive, detecting down to one copy of RNA per microliter, it requires specialized equipment, a runtime of several hours and a centralized laboratory facility. As a result, testing typically takes at least one to two days.
The research team at the labs of Jennifer Doudna, David Savage and Patrick Hsu at the University of California, Berkeley, aims to develop a diagnostic test that is much faster and easier to deploy than qRT-PCR. It has now combined two different types of CRISPR enzymes to create an assay that can detect small amounts of viral RNA in less than an hour.
While the new technique is not yet at the stage where it rivals the sensitivity of qRT-PCR, which can detect just a few copies of the virus per microliter of liquid, it is already able to pick up levels of viral RNA -- about 30 copies per microliter -- sufficient to be used to surveil the population and limit the spread of infections.
"You don't need the sensitivity of PCR to basically catch and diagnose COVID-19 in the community, if the test's convenient enough and fast enough," said co-author David Savage, professor of molecular and cell biology. "Our hope was to drive the biochemistry as far as possible to the point where you could imagine a very convenient format in a setting where you can get tested every day, say, at the entrance to work."
Several CRISPR-based assays have been authorized for emergency use by the Food and Drug Administration, but all require an initial step in which the viral RNA is amplified so that the detection signal -- which involves release of a fluorescent molecule that glows under blue light -- is bright enough to see. While this initial amplification increases the test's sensitivity to a similar level as qRT-PCR, it also introduces steps that make the test more difficult to carry out outside of a laboratory.
The UC Berkeley-led team sought to reach a useful sensitivity and speed without sacrificing the simplicity of the assay.
"For point of care applications, you want to have a rapid response so that people can quickly know if they're infected or not, before you get on a flight, for example, or go visit relatives," said team leader Tina Liu, a research scientist in Doudna's lab at the Innovative Genomics Institute (IGI), a CRISPR-focused center involving UC Berkeley and UC San Francisco scientists.
Aside from having an added step, another disadvantage of initial amplification is that, because it makes billions of copies of viral RNA, there is a greater chance of cross-contamination across patient samples. The new technique developed by the team flips this around and instead boosts the fluorescent signal, eliminating a major source of cross-contamination.
The amplification-free technique, which they term Fast Integrated Nuclease Detection In Tandem (FIND-IT), could enable quick and inexpensive diagnostic tests for many other infectious diseases.
"While we did start this project for the express purpose of impacting COVID-19, I think this particular technique could be applicable to more than just this pandemic because, ultimately, CRISPR is programable," Liu said.
Liu added, "So, you could load the CRISPR enzyme with a sequence targeting flu virus or HIV virus or any type of RNA virus, and the system has the potential to work in the same way. This paper really establishes that this biochemistry is a simpler way to detect RNA and has the capability to detect that RNA in a sensitive and fast time frame that could be amenable for future applications in point of care diagnostics."
The researchers are currently in the process of building such a diagnostic using FIND-IT, which would include steps to collect and process samples and to run the assay on a compact microfluidic device. (ANI)