Despite the impressively rapid development of diagnostic tests for SARS-CoV-2 over the past year and a half. The vast majority of patient samples still have to be sent to a laboratory for processing. Which slows down the pace of COVID-19 case tracking.
However, this could be about to change
Researchers at the Wyss Institute for Biologically Inspired Engineering at Harvard University. The Massachusetts Institute of Technology (MIT) and several Boston-area hospitals. They have created an inexpensive CRISPR-based diagnostic test that allows users to be tested for SARS-CoV-2 and multiple variants of the virus using a sample of their saliva at home. The above, without the need for additional instrumentation.
The diagnostic device, called a minimally instrumented SHERLOCK (miSHERLOCK). It is easy to use and provides results that can be read and verified by an attached smartphone app in one hour.
It successfully distinguished between three different variants of SARS-CoV-2 in experiments and can be quickly reconfigured to detect additional variants such as Delta. The device can be assembled using a 3D printer and commonly available components.
The “MiSHERLOCK” test arrives
“MiSHERLOCK eliminates the need to transport patient samples to a centralized testing location. In addition, it greatly simplifies the sample preparation steps. Giving patients and physicians a faster and more accurate picture of individual and community health, which is critical during an evolving pandemic ”. So said co-first author Helena de Puig, a postdoctoral fellow at the Wyss Institute and MIT.
For the SARS-CoV-2 detection portion of their diagnosis, the group turned to a CRISPR-based technology created in the laboratory of Wyss Core faculty member and lead author of the paper Jim Collins, Ph.D. termed “specific high sensitivity enzymatic reporter unblocking” (SHERLOCK).
How does it work?
SHERLOCK uses CRISPR’s “molecular scissors” to cut DNA or RNA at specific locations. This, with an added advantage: by recognizing your target sequence, This specific type of scissors also cuts other pieces of DNA in the surrounding area. Allowing its engineering to produce a signal indicating that the target has been successfully cut off.
Action mechanisms
The researchers created a SHERLOCK reaction designed to cut SARS-CoV-2 RNA in a specific region of a gene called a nucleoprotein that is conserved in multiple variants of the virus. When molecular scissors, an enzyme called Cas12a, successfully binds to the nucleoprotein gene and cuts it, the single-stranded DNA probes are cut as well. Which produces a fluorescent signal.
They also created additional SHERLOCK assays designed to target a panel of viral mutations in Spike protein sequences that represent three genetic variants of SARS-CoV-2: Alpha, Beta, and Gamma.
Why did the team choose to use saliva?
“When a sample is analyzed for nucleic acids [como ADN o ARN], there are many steps that must be followed to prepare the sample. So that you can actually extract and amplify those nucleic acids.
You must protect the sample while it is in transit to the test center and also ensure that it is not infectious if it is a communicable disease. In order to make this a really easy-to-use diagnostic test, it was important for us to simplify that as much as possible. ” Said co-first author Xiao Tan, MD, Ph.D., clinical fellow at the Wyss Institute and Instructor of Medicine in Gastroenterology at Massachusetts General Hospital.
The team chose to use saliva rather than nasopharyngeal swab samples as diagnostic source material. Why it is easier for users to collect saliva, and studies have shown that SARS-CoV-2 is detectable in saliva for a greater number of days after infection. But raw saliva presents its own challenges: It contains enzymes that break down various molecules, producing a high false-positive rate.
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