With the delta variant having devastating effects on unvaccinated populations and increasing COVID-19 cases worldwide, the pandemic is far from over. Despite the impressively rapid development of SARSCoV2 diagnostic tests over the past year and a half, the vast majority of patient samples still have to be sent to a laboratory for processing, which is slowing the pace of COVID-19 case tracking. If a sample is to be tested for a specific variant of the virus, it must be genetically sequenced, which requires even more time & resources.
Now researchers from the Wyss Institute for Biologically Inspired Engineering at Harvard University, the Massachusetts Institute of Technology (MIT), and several Boston area hospitals have developed a low-cost CRISPR-based diagnostic test that allows users to test for SARSCoV2 and several variants of. get tested the virus. Use a saliva sample at home without the need for additional instruments.
The diagnostic device known as minimally instrumented SHERLOCK (miSHERLOCK) is easy to use and delivers results within an hour that can be read out and verified by a connected smartphone app. It has successfully differentiated between three different variants of SARSCoV2 in experiments and can be quickly reconfigured to recognize additional variants such as Delta. The device can be assembled with a 3D printer and Components generally cost around $ 15, and reusing the hardware brings the cost of individual tests down to $ 6 each.
“MiSHERLOCK eliminates the need to transport patient samples to a central test site and greatly simplifies the sample preparation steps, To provide patients and clinicians with a faster and more accurate picture of individual and community health, which is critical during an evolving pandemic, ”said co-lead author Helena de Puig, Ph.D, Postdoctoral fellow at Wyss Institute and MIT.
The diagnostic device is described in an article published today in Science Advances.
From Supply Chain To SHERLOCK
As a pediatric educator specializing in infectious diseases at Boston Children’s Hospital, co-author Rose Lee, M.D has been on the forefront of the COVID-19 pandemic for over a year. His experience in the clinic served as inspiration for the project that would eventually become mySHERLOCK.
“Simple things that used to be ubiquitous in the hospital, like nasopharyngeal swabs, were suddenly hard to come by, so routine sample processing was interrupted, which is a big problem in a pandemic,” said Lee, who is also a visiting member . at the Wyss Institute. “The motivation of our team for this project was to eliminate these bottlenecks and provide an accurate diagnosis for COVID-19 with less dependence on global supply chains and also to precisely identify the emerging variants.
For the SARSCoV2 detection portion of their diagnosis, the group turned to a CRISPR-based technology developed in the lab of Wyss Core Faculty member and lead author of the article, Jim Collins, Ph.D referred as Specific High Sensitivity Enzymatic Reporter Unlocking”(SHERLOCK). SHERLOCK uses CRISPR’s “molecular scissors” to cut DNA or RNA at specific locations, with an additional advantage: These special types of scissors also cut other pieces of DNA in the area, allowing them to be made from nucleic acid Probing molecules to produce a signal indicating that the target was successfully cut.
The researchers developed a SHERLOCK reaction to cut the SARSCoV2 RNA in a specific region of a gene called nucleoprotein, which is conserved in several variants of the virus. When molecular scissors, an enzyme called Cas12a, The nucleoprotein gene binds and is successfully cleaved, and the single-stranded DNA probe is also cleaved, generating a fluorescent signal. They also developed an additional SHERLOCK test for a series of viral mutations in the spike protein sequence representing the three genetic variants of SARSCoV2: α, β, and γ.
Armed with assays that could reliably detect viral RNA within the accepted concentration range for FDA-cleared diagnostic tests, the team focused on solving arguably the most difficult diagnostic challenge: preparing samples.
Spit, Wait, Scan
“When you test a sample for nucleic acids [like DNA or RNA], there are many steps you have to take to prepare the sample so that you can extract and amplify those nucleic acids. You need to protect the sample while it is in transit to the testing center and make sure it is not infectious if you are dealing with a communicable disease, and to make this a really easy-to-use diagnostic test it was important to us to simplify that as much as possible, “said co-author Xiao Tan, M., Ph.D, Clinical Fellow at the Wyss Institute and Instructor of Medicine in Gastroenterology at Massachusetts General Hospital.
The team chose to use saliva instead of nasopharyngeal swab samples as the collection method because it is easier for users to collect saliva, and studies have shown that SARSCoV2 is detectable in saliva for a longer number of days after infection. But raw saliva presents its own challenges. – Contains enzymes that break down various molecules & generate a high-rate false-positives.
The researchers developed a novel technique to solve this problem: first, they added two chemicals called DTT and EGTA to the saliva and heated the sample to 95 ° C for 3 minutes, which removed the false positive signal from the untreated saliva & slice open any virus particles. They then inserted a porous membrane designed to capture RNA on its surface, which could ultimately be added directly to the SHERLOCK reaction to produce a result.
To integrate saliva sample preparation and the SHERLOCK reaction into a diagnosis, the team designed a simple battery-operated device with two chambers: a heated sample preparation chamber and an unheated reaction chamber. A user spits into the sample preparation chamber, turns on the heat, & waits 3-6 min for the saliva to wicked the filter. The user removes the filter & transfer it in the column of the reaction chamber, Then pushes a plunger, which sets the filter into the chamber and pierces a reservoir of water to activate the SHERLOCK reaction. 55 minutes later, the user looks into the reaction chamber through the window of the tinted transilluminator window & confirms the presence of a fluorescent signal. You can also use a smartphone app that analyzes the pixels that the smartphone camera records to make a clear positive or negative diagnosis.
The researchers tested their diagnostic device on clinical saliva samples from 27 COVID-19 patients and 21 healthy patients and found that miSHERLOCK correctly identified COVID-19-positive patients in 96% of time & patients without the disease in 95% of time. They also tested its performance against the SARSCoV2 variants Alpha, Beta, and Gamma by adding full-length synthetic viral RNA to healthy human saliva containing the mutations that each variant contained, and found the device to be effective at a variety of viral RNA concentrations.
“One of the best things about miSHERLOCK is that it is completely modular. The device itself is separate from assays so you can join different assays for the specific RNA or DNA sequence you want to detect, ”said co-author Devora. Najjar, research fellow at the MIT Media Lab and the Collins Lab. “The device costs about $ 15, but mass production would reduce the case to about $ 3. Assays for new targets can be created in about 2 weeks, allowing rapid development of tests for new variants of COVID-19 & other diseases.
Ready For Real World
The miSHERLOCK team designed their device with low resource settings in mind as the pandemic exposed the great inequalities in access to health care between different parts of the world. The device’s hardware can be built by anyone with access to a 3D printer, and the files & circuit designs are all publicly available online. The addition of a smartphone app was also intended for environments with limited resources, as the cellular service is available virtually anywhere in the world, even in areas that are difficult to reach on foot. The team is committed to working with manufacturers who are interested in producing miSHERLOCK on a large scale for worldwide distribution.
“When the miSHERLOCK project began, there was almost no monitoring for SARSCoV2 variants. We knew variant tracking would be incredibly important by assessing the long-term impact of COVID-19 on local and global communities, we strive to create a truly decentralized, flexible and easy-to-use diagnostic platform, “said Collins, who is also a Termeer Professor of Medical Engineering and Science at MIT “By solving the sample preparation problem, we have ensured this device is virtually-ready to use and we are excited to be working with industry partners to make it commercially available.
“By combining cutting-edge biotechnology with inexpensive materials, this team has created a powerful diagnostic tool that can be manufactured and used in local area by people without advanced medical degrees. It’s a perfect example of the Wyss Institute’s mission in action: putting life-changing innovations in the hands of the people who need them, ”said Wyss Founding Director Don Ingber, M., Ph., Who is also a professor. Judah Folkman Professor of Vascular Biology at Harvard Medical School and Boston Children’s Hospital, and Professor of Bioengineering at Harvard John A. Paulson School of Engineering & Applied Sciences.
The research were published on Science Advances.