The pandemic is not yet over, as the Delta variant has wreaked havoc on unvaccinated populations and the number of COVID-19 cases has skyrocketed around the world. Despite the surprisingly rapid development of diagnostic tests for SARS-CoV-2 over the past year and a half, most patient samples need to be sent to the lab for processing, slowing the pace of tracking COVID- cases. 19. He became. If you want to test your sample for a specific variant of the virus, you have to sequence it, which takes even more time and resources.
Currently, researchers at Harvard’s Wyss Institute for Biologically Inspired Engineering, the Massachusetts Institute of Technology (MIT), and several regional hospitals in Boston are using inexpensive CRISPR-based diagnostic tests that allow users to test themselves for SARS-CoV. is created. -Two and multiple variants of the virus that use saliva samples at home. No additional equipment is required.
A diagnostic device called SHERLOCK with minimal instrumentation (miSHERLOCK) is easy to use and provides results that can be read and verified within an hour with the included smartphone app. Experiments can successfully distinguish between the three different variants of SARS-CoV-2 and quickly reconfigure themselves to detect additional variants such as deltas. The device can be assembled using a 3D printer and commonly available components for around $ 15, and reusing the hardware reduces the cost of each test to $ 6 each.
“MiSHERLOCK eliminates the need to transport patient samples to a centralized testing site, dramatically simplifies sample preparation procedures, and provides patients and physicians with faster, more accurate images of personal and community health. First author, Dr. Helena de Puig, Wyss Institute for Bis, and postdoctoral researcher at MIT.
The diagnostic device is described in an article published today. Scientists progress..
From the supply chain to SHERLOCK
As a pediatric instructor at Children’s Hospital in Boston, he specializes in infectious diseases. Co-lead author Dr Roseley has been at the forefront of the COVID-19 pandemic for over a year. His experience at the clinic inspired the project that eventually became miSHERLOCK.
“A simple thing that was once all over the hospital, like a nasopharyngeal swab, suddenly became hard to find, interrupting routine sample processing procedures,” said Lee, a visiting researcher. This is a big problem in the situation. ”At the Institut Wis. “Our team’s motivation for this project was to remove these bottlenecks, reduce reliance on the global supply chain, and provide an accurate diagnosis of COVID-19 and the variants that are starting to appear. emerge. Could also be detected accurately. “
For the SARS-CoV-2 detection portion of their diagnosis, the group is a member of the Wyss Core Faculty and lead author Jim Collins, Ph.D. We turned to the CRISPR-based technology created in our lab. This is called the “Unlocking of the specific sensitive enzyme reporter” (SHERLOCK). SHERLOCK uses CRISPR’s “molecular scissors” to cut DNA or RNA at specific locations. In addition, this particular type of scissors also cleaves other parts of the surrounding DNA, allowing it to be manipulated using a nucleic acid probe molecule. Generates a signal indicating that the target has been successfully disconnected.
The researchers created a SHERLOCK reaction designed to cleave SARS-CoV-2 RNA into specific regions of a gene called a nucleoprotein that is conserved across several mutants of the virus. When a molecular scissor (an enzyme called Cas12a) successfully binds and cleaves a nucleoprotein gene, the single-stranded DNA probe is also cleaved and a fluorescent signal is generated. They also created an additional SHERLOCK assay designed to target a panel of viral mutations in peplomeric protein sequences representing three genetic variants of SARS-CoV-2 (alpha, beta, gamma).
The team then focused on solving sample preparation, perhaps the most difficult diagnostic challenge, with an assay that could reliably detect viral RNA within the concentration range accepted by the assays. diagnostic tools certified by the FDA.
Spit, wait, scan
“When you test nucleic acid samples [like DNA or RNA]Many steps are required to prepare the sample so that you can actually extract and amplify these nucleic acids. Samples should be protected during transport to the laboratory. Also, if you are dealing with an infectious disease, you need to make sure that it is not infectious. To make this test really easy to use, it was important to make it as simple as possible, ”said Xiao Tan, MD, PhD, clinical researcher and gastroenterology instructor at the Wyss Institute at Massachusetts General Hospital. Said.
The team chose to use saliva instead of the nasopharyngeal swab sample as the collection method. This is because studies have shown that it is easier for users to collect saliva and that SARS-CoV-2 can be detected in saliva for more days after infection. However, untreated saliva has its own challenges. It contains enzymes that break down various molecules, resulting in a high rate of false positives.
Researchers have developed new technologies to solve the problem. First, two chemicals, DTT and EGTA, were added to the saliva and the sample was heated at 95 ° C for 3 minutes. This removed the false positive signal from the untreated saliva and sliced and opened the virus particles. We could then incorporate a porous membrane designed to trap RNA on the surface and finally add it directly to the SHERLOCK reaction to produce results.
To integrate the saliva sample preparation and SHERLOCK reaction into a single diagnostic, the team designed a simple battery-powered device with two chambers (a heated sample preparation chamber and an unheated reaction chamber). The user spits into the sample preparation chamber, turns it on, and waits 3-6 minutes for saliva to be drawn into the filter. The user removes the filter, transfers it to the column of the reaction chamber, pushes the piston to put the filter in the chamber, pierces the cistern and activates the SHERLOCK reaction. After 55 minutes, the user examines the reaction chamber through a colored transilluminator window to confirm the presence of fluorescent signals. You can also use the included smartphone app to analyze the pixels recorded in your smartphone’s camera to provide a clear positive or negative diagnosis.
Researchers tested diagnostic equipment using clinical saliva samples from 27 COVID-19 patients and 21 healthy patients, with a 96% probability of miSHERLOCK in COVID-19 positive patients and 95%. We have found that we have a good chance of correctly identifying a patient without disease. They also tested the performance against the alpha, beta and gamma mutants of SARS-CoV-2 by full-length synthetic viral RNA containing mutations representing each mutant in healthy human saliva, and the device was found to be effective. across the range of viruses. RNA. concentration.
“One of the advantages of miSHERLOCK is that it is completely modular. The device itself is isolated from the test, so you can run different tests for the specific sequence of RNA or DNA you are trying to detect. You can plug it in, ”says co-first author Devora Najjar, MIT Media Lab and Collins Lab Research Assistant. “The device costs around $ 15, but mass production makes the case around $ 3. A new target test can be created in about two weeks, testing new variants of COVID-19 and other diseases. It can be developed quickly. “
Prepare for the real world
The pandemic has revealed significant inequalities in access to medical care in different parts of the world, which is why the miSHERLOCK team created the device with scarce resources in mind. Device hardware can be built by anyone with access to a 3D printer, and all files and circuit designs are posted online. The addition of smartphone apps was intended for resource-constrained environments, as mobile phone services can be used virtually anywhere in the world, even in areas that are difficult to reach on foot. The team looks forward to working with manufacturers interested in producing miSHERLOCK on a large scale for worldwide distribution.
“When the miSHERLOCK project was started, there was little surveillance for SARS-CoV-2 variants. Tracking variants is very important when assessing the long-term impact of COVID-19 on local and global communities. I knew it would be important to me. I pushed myself to create a truly decentralized, flexible and user-friendly diagnostic platform, ”said Professor Termeer of Biomedical Engineering and Science at MIT. said a Collins. “In solving the sample It’s a matter of preparation. We have confirmed that this device is ready for use by consumers. We are excited to be working with our industry partners to bring it to market. “
“By combining cutting edge biotechnology with low cost materials, the team is powerful. Diagnostic device It can be manufactured and used locally by people without a high medical qualification. It is a perfect example of the mission of the Wis Institute. Putting life-changing innovations in the hands of those who need them, ”said Dr. Don Ingbar, MD, Founding Director of Wis, who is also Professor Judah Folkman. Professor of vascular biology at Harvard Medical School and Boston Children’s Hospital, and of bioengineering at the John A. Paulson School of Applied Sciences at Harvard University.
Face mask that can diagnose COVID-19
Minimized SHERLOCK (miSHERLOCK) for CRISPR-based point-of-care diagnosis of SARS-CoV-2 and novel mutants, Scientists progress (2021). Advances.sciencemag.org/lookup… .1126 / sciadv.abh2944
Quote: Https: //medicalxpress.com/news/2021-08-covid-variants.html Test for the detection of the COVID-19 variant in saliva (August 6, 2021) obtained on August 6, 2021
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