A team of researchers from MIT and Harvard University have designed a simple, inexpensive device that can detect and identify multiple variants of SARS-CoV-2 in a saliva sample in about an hour. The small, tabletop device, which the researchers dubbed miSHERLOCK (minimally instrumented SHERLOCK, after the CRISPR-based DNA-editing tool SHERLOCK), provides the same accuracy as a PCR test.
‘Our goal was to create an entirely self-contained diagnostic that requires no other equipment,’ said Xiao Tan, a clinical fellow at Harvard’s Wyss Institute. ‘Essentially the patient spits into this device, and then you push down a plunger and you get an answer an hour later.’
Capable of being assembled using a 3D printer and commonly available components that cost a total of about US$15, the device utilises an RNA guide strand that allows detection of specific target RNA sequences, as well as CRISPR/Cas enzymes that cleave those sequences and produce a fluorescent signal. All of the device’s molecular components can be freeze-dried for long-term storage and then reactivated when exposed to water.
Creating a saliva test required the incorporation of a method for disabling salivary nucleases, which destroy nucleic acids such as RNA, potentially leading to a high rate of false positives. The researchers overcame this problem by using heat and two chemical reagents to inactivate the nucleases. The viral RNA is then extracted and concentrated by passing the saliva through a membrane.
‘That membrane was key to collecting the nucleic acids and concentrating them so that we can get the sensitivity that we are showing with this diagnostic,’ said Rose Lee, a visiting fellow at the Wyss Institute.
The RNA sample is then exposed to freeze-dried CRISPR components, which are activated when sealed water packets within the device are punctured. The one-pot reaction then amplifies the RNA sample and detects the target RNA sequence if it’s present. The device can have up to four modules that each target a different RNA sequence – one that can detect any strain of SARS-CoV-2 and others that are specific to mutations associated with some of the currently known variants.
‘One of the great things about miSHERLOCK is that it’s entirely modular. The device itself is separate from the assays, so you can plug in different assays for the specific sequence of RNA or DNA you’re trying to detect,’ said Devora Najjar, a research assistant at MIT. ‘Assays for new targets can be created in about two weeks, enabling the rapid development of tests for new variants of Covid-19 and other diseases.’
‘The ability to detect and track these variants is essential to effective public health, but unfortunately, variants are currently diagnosed only by nucleic acid sequencing at specialised epidemiological centres that are scarce even in resource-rich nations,’ said Helena de Puig, a postdoc at the Wyss Institute, who led the study.
Once the test is complete, the fluorescent readout generated by the assay is analysed by accompanying smartphone app, which gives the user a clear positive or negative result and sends that result to the relevant public health department.
The diagnostic device is described in a paper published in Science Advances.