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Test to detect COVID-19 mutation in saliva

miSHERLOCK is a self-contained home diagnostic platform that allows users to test their saliva for the presence of SARS-CoV-2 and distinguish between multiple variants of the virus. Credit: Harvard University Wyss Institute

The pandemic is not over yet, as the Delta variant has caused havoc in unvaccinated populations and the number of cases of COVID-19 has skyrocketed around the world. Despite the surprisingly rapid development of SARS-CoV-2 diagnostic tests over the past year and a half, most patient samples have to be sent to the lab for processing, slowing the pace of COVID-19 case follow-up. It has become. If you want to test your sample for a specific variant of the virus, you need to sequence it, which takes even more time and resources.

Currently, researchers at Harvard’s Wyss Institute for Biologically Inspired Engineering, Massachusetts Institute of Technology (MIT), and several Boston regional hospitals are using cheap 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 Minimally Instrumented SHERLOCK (miSHERLOCK) is easy to use and provides results that can be read and checked within an hour with the included smartphone app. Experiments can successfully distinguish between the three different variants of SARS-CoV-2 and quickly reconfigure to detect additional variants such as deltas. The device can be assembled using a 3D printer and commonly available components for about $ 15, and reusing the hardware reduces the cost of each assay to $ 6 each.

“MiSHERLOCK eliminates the need to transport patient samples to a centralized test site, greatly 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 Postdoc researcher at MIT.

The diagnostic device is described in a paper published today. Science Advances..

From supply chain to SHERLOCK

As a pediatrics 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. Her experience at the clinic inspired the project that eventually became miSHERLOCK.

“A simple thing that was once everywhere in the hospital, like a nasopharyngeal swab, suddenly became hard to come by, interrupting routine sample processing procedures,” said Lee, a visiting scholar. It’s a big problem in the situation. ” At the Wis Institute. “Our team’s motivation for this project was to eliminate these bottlenecks, reduce reliance on the global supply chain and provide an accurate diagnosis of COVID-19, and variants that are beginning to emerge. Was also able to be detected accurately. “

Test to detect COVID-19 mutation in saliva

In the diagnostic procedure, the user simply spits into the sample preparation chamber, moves the collection disc to the reaction chamber and pushes the plunger to activate the reaction and minimize the risk of mutual contamination. Credit: Harvard University Wyss Institute

For the SARS-CoV-2 detection portion of their diagnosis, the group is a member of the Wyss Core Faculty and senior author Jim Collins, Ph.D. We turned to CRISPR-based technology created in our lab. It is called “Specific Sensitive Enzyme Reporter Unlock” (SHERLOCK). SHERLOCK uses CRISPR’s “molecular scissors” to cut out 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 that the target has been successfully disconnected.

Researchers have created a SHERLOCK reaction designed to cleave SARS-CoV-2 RNA at specific regions of a gene called a nucleoprotein that is conserved across multiple mutants of the virus. When a molecular scissors (an enzyme called Cas12a) successfully binds to 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 peplomer protein sequences representing three SARS-CoV-2 genetic variants (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 FDA-certified diagnostic tests.

Spit, wait, scan

“When testing sample nucleic acids [like DNA or RNA], There are many steps required to prepare the sample so that you can actually extract and amplify those nucleic acids. Samples must 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 really easy to use Diagnostic testIt was important to simplify it as much as possible, “said Xiao Tan, MD, Ph.D., a clinical fellow and gastroenterologist instructor at the Wyss Institute at Massachusetts General Hospital. Says.

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 false positive rate.

Researchers have developed new technologies to solve the problem. First, two chemicals, DTT and EGTA, were added to 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 saliva sample preparation and the SHERLOCK reaction into a single diagnosis, the team designed a simple battery-powered device with two chambers (a heated sample preparation chamber and a non-heated reaction chamber). The user spits into the sample preparation chamber, ignites it, and waits 3-6 minutes for saliva to be sucked into the filter. The user removes the filter, transfers it to the reaction chamber column, pushes the plunger to put the filter into the chamber, punctures 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 registered in your smartphone’s camera to provide a clear positive or negative diagnosis.

Test to detect COVID-19 mutation in saliva

The included smartphone app analyzes the fluorescence readings generated by the assay and provides the user with clear “positive” or “negative” results. The results can also be shared with doctors and health organizations to track the spread of the disease. Credit: Harvard University Wyss Institute

Researchers tested diagnostic equipment using clinical saliva samples from 27 COVID-19 patients and 21 healthy patients, with a 96% chance of miSHERLOCK in COVID-19-positive patients and 95%. We found that we had a good chance of correctly identifying a disease-free patient. They also tested performance against alpha, beta, and gamma SARS-CoV-2 mutants by spiked full-length synthetic viral RNA containing mutations representing each mutant into healthy human saliva, and the device It was found to be effective over the entire range of viral RNA. concentration.

“One of the great things about miSHERLOCK is that it’s fully modular. The device itself is isolated from the assay, so you can do different assays for the specific sequence of RNA or DNA you’re 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 about $ 15, but mass production makes the housing about $ 3. A new target assay can be created in about two weeks, testing new variants of COVID-19 and other diseases. It can be developed quickly. “

Preparing for the real world

The pandemic revealed significant inequality in medical access in different parts of the world, so the miSHERLOCK team created the device with low resource settings in mind. The hardware of the device can be built by anyone with access to a 3D printer, and all files and circuit designs are published online. The addition of smartphone apps was intended for resource-constrained settings, as mobile phone services can be used virtually anywhere in the world, even in areas that are difficult to reach on foot. The team is eager to work with manufacturers interested in producing miSHERLOCK on a large scale for global distribution.

“When the miSHERLOCK project was started, there was little monitoring of SARS-CoV-2 variants. Variant tracking is very important when assessing the long-term impact of COVID-19 on the local and global communities. I knew it would be important for 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. One Collins said. “By solving sample It’s a matter of preparation. We have confirmed that this device is ready for use by consumers. We are excited to work with our industry partners to bring it to market. “

“By combining state-of-the-art biotechnology with low-cost materials, the team is powerful. Diagnostic device It can be manufactured and used at the local level by people without a high degree of medical degree. This 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 Bioengineering at Harvard University John A. Paulson School of Applied Sciences.


Face mask that can diagnose COVID-19


For more information:
Minimalized SHERLOCK (miSHERLOCK) for CRISPR-based point-of-care diagnosis of SARS-CoV-2 and new mutants, Science Advances (2021). Advances.sciencemag.org/lookup… .1126 / sciadv.abh2944

Provided by
Harvard University

Quote: Https: //medicalxpress.com/news/2021-08-covid-variants.html Test to detect COVID-19 variant in saliva (August 6, 2021) obtained on August 6, 2021

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Test to detect COVID-19 mutation in saliva

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