Five minutes
A CRISPR/Cas-based coronavirus test will give a result in five minutes
Polina Loseva, N+1
A new test for SARS-CoV-2 based on the CRISPR/Cas system promises a reliable result in five minutes. At the same time, it detects even dozens of copies of viral RNA in a microliter of the sample and gives not only a quantitative, but also a qualitative response. To achieve this result, scientists used a set of several RNA guides instead of one, and adapted a smartphone camera as a signal detector. The results of the work were previously published on the portal of non-licensed manuscripts medRxiv (Fozouni et al., Direct detection of SARS-CoV-2 using CRISPR-Cas13a and a mobile phone).
Drawings from the article in medRxiv.
Despite the fact that the first tests for SARS-CoV-2 appeared in a matter of weeks after the outbreak, it is still difficult to identify infected people in time. The problem is that the tests should be massive and fast, because some people who may be contagious do not have symptoms or will develop them only after a few days, and the concentration of the virus in their saliva often does not differ from that of COVID-19 patients.
The most common way to catch a virus in a sputum smear is real–time PCR (polymerase chain reaction). The principle of its operation is that a set of enzymes copies each molecule of viral RNA and marks it with a fluorescent dye – and this happens until the light signal becomes strong enough to be recognized by the device.
The PCR method is popular mainly due to its sensitivity – it allows you to "see" a single copy of RNA in a microliter of the sample. But it has its drawbacks: the test takes from several tens of minutes to several hours, which, taking into account logistical difficulties, turns into at least a day of waiting for the result, during which the asymptomatic carrier manages to spread the infection further.
Therefore, there was a request for faster tests, which do not necessarily have to be so sensitive: according to clinical studies, with an RNA concentration below a thousand copies per microliter, the number of viral particles in the smear is no longer so significant and does not pose a great danger. One of the alternative methods that could speed up testing was the CRISPR/Cas system (the one for which they recently awarded Nobel Prize in Chemistry).
To search for SARS-CoV-2, this system was adapted in the spring, and it worked like this: RNA is quickly extracted from the patient's smear and copied in its entirety several times. Then a guide RNA complementary to the virus sequences is added. This RNA binds to the viral genome and activates an enzyme of the Cas group, which begins to cut viral RNA – and at the same time all single-stranded RNA molecules that it meets. If an RNA molecule bound to a dye is added to the solution, then during cleavage it will be released and give a color signal that the virus has been detected in the sample.
The whole procedure took about an hour – which is already faster than real–time PCR, but still not fast enough for mass testing, and most importantly, it requires complex equipment and reagents. Therefore, a group of scientists from the University of California, led by Nobel laureate Jennifer Doudna, began developing an even faster and simpler test for SARS-CoV-2. The researchers created a dozen guide RNAs (all of them were complementary to different parts of the viral genome) and selected the most effective pair from them. The system, in which there were two RNA guides, made it possible to solve several problems at once.
The principle of operation of the CRISPR/Cas-based test with two guide RNAs.
Firstly, each copy of the viral RNA triggers the work of not one enzyme, Cas13a, but several at once. And this, in turn, allows you to do without first copying the entire RNA in the sample – the signal is already strong enough. In the full version, the new test takes half an hour, but after the first five minutes of observation, scientists were able to unambiguously determine which samples are positive and which are negative.
Secondly, now the test system works even more accurately and does not respond to the RNA of other viruses: the researchers tested it on several related SARS-CoV-2 coronaviruses (including MERS) and did not receive a signal. At the same time, the sensitivity remained acceptable – about a hundred viral RNAs per microliter. When scientists tried to add a third guide RNA to the system, the sensitivity was even higher – up to 31 copies per microliter.
Thirdly, now the test allows you to evaluate not only the presence, but also the amount of viral RNA. Since the RNA copying stage has disappeared from the method, the number of RNA copies in the sample does not change, and it can be judged by the signal intensity. Researchers believe that such technology could help track the development of the disease in individual patients.
Finally, as a detector, the scientists used a smartphone camera, on the basis of which they constructed a laser fluorescent microscope, adding a laser beam source and a filter. It turned out that the sensitivity of the camera is enough to do without bulky devices and distinguish signals of different intensity from the sample.
The principle of operation of a smartphone-based microscope.
The new system so far works only in laboratory conditions, but Jennifer Dudna told the magazine Science that her colleagues are looking for a way to bring their invention to market. They believe that their method will remain relevant for a long time: even if a coronavirus vaccine appears in the coming months, there is no guarantee yet that the immune response caused by it will be long–term - which means there will be a need for mass testing to track local outbreaks of infection.
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