PCR tests are considered the gold standard for the detection of Sars-CoV-2, but they are complex and require a certain medical infrastructure. Researchers have now developed a possible alternative: their miSHERLOCK test system should detect Covid-19 using saliva samples within just one hour and at the same time identify variants of the virus. The device is designed in such a way that laypeople can use it at home and read the result via a smartphone app. The cost-effective production should also make it accessible to resource-poor areas.
An important component in containing the Covid-19 pandemic is a broad-based test strategy that reliably identifies infections with Sars-CoV-2 and, in particular, controls the spread of variants. However, especially in countries with a weak medical infrastructure, reliable tests are often not available, and test capacities are limited even in industrialized countries. PCR tests, the current gold standard for detecting the virus, require medically trained staff and laboratories in which the samples can be evaluated. The simple and cheaper rapid antigen tests are less sensitive and cannot identify virus variants.
Fluorescence shows positive result
A team led by Helena de Puig from Harvard University in Boston has now designed a test system that detects Sars-CoV-2 in saliva samples – and is supposed to be just as reliable as a PCR test, but faster, cheaper and less complicated. The modular system also already includes tests for various known variants of the virus. “We have shown that our platform can be programmed in such a way that it recognizes newly occurring variants and that we can repurpose it very quickly,” says de Puig’s colleague James Collins. “In this study we focused on the British, South African and Brazilian variants, but the diagnostic platform could easily be adapted to the Delta variant and other emerging variants.”
The test is based on the so-called SHERLOCK technology (specific high-sensitivity enzymatic reporter unlocking), which in turn uses the Crispr / Cas gene scissors. Special Cas enzymes are used that generate a fluorescent signal when they recognize and cut the viral genetic material. An RNA guide strand determines which sequence the Cas enzymes react to. The device from de Puig and her colleagues contains several sample chambers, each with different guide strands. While the Cas enzymes in one of the sample vessels bind to a sequence that is common to all variants of Sars-CoV-2, in the other sample vessels they react to specific sequences of the virus mutations and can thus prove whether a variant is present.
Easy to use for laypeople
To make it as easy as possible for the user to use, the researchers decided to use saliva samples as the starting material. If there is an infection, the saliva contains a lot of virus material and is therefore well suited for detection. However, since other components of saliva, so-called salivary nucleases, can produce false positive test results even without infection, the researchers included a crucial preparatory step: When the user inserts the sample into the device, it is first heated to 95 degrees Celsius for three minutes and mixed with two chemicals that deactivate the salivary nucleases. The saliva is then passed through a membrane that extracts the viral RNA. “This membrane was the key to collecting and concentrating the nucleic acids so that we can achieve the sensitivity that we need for this diagnostic,” says de Puig’s colleague Rose Lee.
After this preparation, the user places the sample in the main chamber of the device, which contains the freeze-dried Crispr / Cas components. He pushes it into the device with a plunger and at the same time pierces sealed water packets that set the reaction in motion. After 55 minutes, he can read the result through a tinted window – fluorescence means that the test is positive. The results can be evaluated with a smartphone app – and, if necessary, transmitted directly to the responsible health authority. “Our goal was to develop a completely independent diagnostic device that does not require any additional devices,” explains de Puig’s colleague Xiao Tan. “Basically, the patient spits into this device, pushes down a plunger and receives an answer an hour later.”
Also for areas with low resources
The researchers have tested their device, which they call the “minimally instrumented SHERLOCK” (miSHERLOCK), with saliva samples from 27 Covid-19 patients and 21 healthy control persons. The test only delivered one false positive and one false negative result – i.e. 96 percent of those infected were identified as infected and 95 percent of those who were healthy as healthy. Tests with saliva samples, to which the researchers added synthetic virus sequences, also provided results that were similarly accurate as PCR tests. The device currently costs about $ 15 each to manufacture. The authors believe that mass production will bring the price of the case down to around three dollars. The enzymatic components are also only a few dollars and would be cheaper if mass produced.
From the authors’ point of view, miSHERLOCK can thus compensate for inequalities in access to health care. Since almost all parts can be produced with the help of a 3D printer, it is easily available even in resource-poor areas. “The motivation of our team for this project was to eliminate bottlenecks and provide accurate diagnostics for Covid-19 without having to rely on global supply chains, and also to precisely identify the variants that are emerging,” says Lee. The researchers are currently striving for approval of the diagnostic agent by the American FDA and are looking for industrial partners who produce miSHERLOCK on a large scale.
Source: Helena de Puig (Harvard University, Boston) et al., Science Advances, doi: 10.1126 / sciadv.abh2944