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Although today’s gold standard for COVID-19 diagnostic testing uses qRT-PCR (quantitative reverse transcriptase-polymerase chain reaction (PCR)) which is very sensitive and detects one copy of RNA per microliter, it requires specialized equipment and running time.
A research team led by scientists in the laboratories of Jennifer Doudna, David Savage, and Patrick Hsu of the University of California, Berkeley, is working to develop a diagnostic test that is faster and easier to deploy than qRT-PCR
Although this new technology has not yet reached the sensitivity of qRT-PCR, which can detect several copies of the virus per microliter, it has been able to reach the level of detection-30 copies per milliliter, which is enough to monitor transmission Infection
"If this test is convenient and fast enough, the sensitivity of PCR will not be needed to basically capture and diagnose COVID-19 in the community," said David Savage, a co-author of the study and professor of molecular and cell biology.
The research results were published online in Nature Chemical Biology on August 5
Between
The FDA urgently authorized several CRISPR-based detection methods, but they all require an initial step, that is, amplification of viral RNA in order to detect the signal
The research team led by the University of California at Berkeley tried to achieve useful sensitivity and speed without sacrificing detection simplicity
"For testing applications, we want to have a quick response so that people can quickly know if they are infected, when you got on the plane, for example, or went to visit relatives," said Tina Liu, the first article of the article
In addition to adding steps, another disadvantage of initial amplification is that because it generates billions of viral RNA copies, there is a greater possibility of cross-contamination of patient samples
This non-amplification technology, which researchers call Fast Integrated Nuclease Detection In Tandem (FIND-IT, Biology Tongzhu), can provide fast and inexpensive diagnostic tests for many other infectious diseases
Researchers are currently using FIND-IT to build diagnostic procedures, which include the steps of collecting and processing samples and running tests on compact microfluidic devices
Tandem Cas protein
In order to remove the amplification step, the team used a CRISPR enzyme, Cas13, to first detect viral RNA and use another Cas protein called Csm6 to amplify the fluorescent signal
Cas13 is a universal scissors for cutting RNA: once it binds to the target sequence specified by the guide RNA, it is ready to cut a wide range of other RNA molecules
The ingenuity of this research is to use Csm6 to amplify the effect of Cas13
In order to facilitate Cas13 detection, the researchers designed a special activator molecule that will be cleaved when Cas13 detects viral RNA
"When Cas13 is activated, it cuts this small activator and removes the fragments that protect it," Liu said.
The team of researchers also integrated an optimized guide RNA combination to enable Cas13 to more sensitively recognize viral RNA
.
When it was used in combination with Csm6 and its activator, the team was able to detect as little as 31 copies of SARS-CoV-2 RNA per microliter in just 20 minutes
.
The researchers also added RNA extracted from patient samples to the FIND-IT analysis in the microfluidic box to see if the analysis is suitable for running on portable devices
.
Using a small device with a camera, they can detect SARS-CoV-2 RNA extracted from patient samples, and its sensitivity can capture COVID-19 infection at its peak
.
"This tandem nuclease method-Cas13 plus Csm6 combines everything into a single reaction at a single temperature of 37°, so it does not require high temperature heating or multiple steps, which is necessary for other diagnostic techniques.
"Liu said, "I think this opens up opportunities for faster and simpler tests that can achieve sensitivity comparable to other current technologies, and may achieve higher sensitivity in the future
.
"
The development of this amplification-free method for RNA detection is due to the repositioning of IGI's internal research when the epidemic began to solve the problem of COVID-19 diagnosis and treatment
.
In the end, five laboratories at the University of California, Berkeley and two laboratories at the University of California, San Francisco participated in this research project, which is one of the many laboratories in IGI
.
“When we started this project, we wanted to create something that was comparable to PCR but did not require amplification-this is our dream,” said Savage, the lead researcher of the project.
“From a sensitivity point of view, we have A gap of about ten thousand times
.
We have done it about a thousand times; we have reduced it by about three orders of magnitude
.
So, we are almost there
.
Last April, when we actually started to draw it out, it seemed almost Impossible
.
" (Biological Communication)
Original reference:
1.
Tina Y.
Liu, Gavin J.
Knott, Dylan CJ Smock, John J.
Desmarais, Sungmin Son, Abdul Bhuiya, Shrutee Jakhanwal, Noam Prywes, Shreeya Agrawal, María Díaz de León Derby, Neil A.
Switz, Maxim Armstrong, Andrew R.
Harris, Emeric J.
Charles, Brittney W.
Thornton, Parinaz Fozouni, Jeffrey Shu, Stephanie I.
Stephens, G.
Renuka Kumar, Chunyu Zhao, Amanda Mok, Anthony T.
Iavarone, Arturo M.
Escajeda, Roger McIntosh, Shineui Kim, Eli J.
Dugan, Jennifer R.
Hamilton, Enrique Lin-Shiao, Elizabeth C.
Stahl, Connor A.
Tsuchida, Erica A.
Moehle, Petros Giannikopoulos, Matthew McElroy, Shana McDevitt, Arielle Zur, Iman Sylvain, Alison Ciling , Madeleine Zhu, Clara Williams, Alisha Baldwin, Katherine S.
Pollard, Ming X.
Tan, Melanie Ott, Daniel A.
Fletcher, Liana F.
Lareau, Patrick D.
Hsu, David F.
Savage, Jennifer A.
Doudna.
Accelerated RNA detection using tandem CRISPR nucleases.
Nature Chemical Biology, 2021; DOI: 10.
1038/s41589-021-00842-2