According to the World Health Organization (WHO), the number of newly diagnosed COVID-19 patients worldwide increased by 1 million in the three days from October 8 to October 10. With the arrival of autumn and winter in the northern hemisphere, controlling the COVID-19 epidemic is still facing serious challenges. Fast and accurate nucleic acid testing helps health workers detect patients infected with the new coronavirus earlier, allowing them to be treated/isolated and contacted.
Most nucleic acid tests today still require samples to be sent to specialized laboratories for processing, often one day or more to obtain results, which can delay the treatment and isolation of patients.
Drug Mingkang content team drawing.
As a result, researchers are working together to develop faster and easier nucleic acid detection. Using CRISPR technology to speed up detection is one of the directions. The Pharmaceutical Mingkang content team has also previously reported on the efforts of the team led by renowned scholar Dr. Zhang Feng and his collaborators to develop simple new coronavirus detection using CRISPR technology. In order to make the detection faster and easier, Zhang Feng’s STOPCovid detection has been iterative, not only using thermostat PCR instead of traditional PCR, but also reducing the process of purifying RNA, but also improving the sensitivity of the detection through optimization steps.
A team led by Dr. Jennifer Doudna, one of this year’s Nobel Laureates in Chemistry, and her partners have published a paper on preprinted website medRxiv, developing a new type of coronavirus detection based on CRISPR-Cas13a technology. What sets this test apart is that it does not require the use of RT-PCR amplification for viral RNA, which directly quantifys the level of viral RNA in a sample. The researchers have also developed a smartphone-based camera detection system that allows medical staff to read test results in environments outside the lab without the need for sophisticated instruments.
Increase the sensitivity of CRISPR detection without PCR.
The core principles of this test are not difficult to understand. The researchers used an enzyme called Cas13a and a combination of CRISPR RNA (crRNA) to form a complex. Cas13a enzymes can be activated when crRNA is combined with specific sequences on viral RNA. Cas13a is an interesting enzyme that, once activated, indiscriminately slices any other single-stranded RNA molecules encountered around it. Using this feature, the researchers added a fluorescent molecule connected by RNA to the sample. Once Cas13a is activated, the RNA on this particular molecule is cut off, releasing fluorescent molecules that emit fluorescence. This allows the presence of virus-specific sequences to be detected by reading fluorescent signals.
CrispR detection, previously based on this principle, amplifies RNA in samples via PCR in order to improve the sensitivity of the test. However, this PCR step not only increases the time required for testing, but also means that the number of tests is limited by the supply of PCR reagents.
CrRNA using multiple virus sequences can improve detection sensitivity.
In the study, the researchers used another method to improve the sensitivity of the test. They speculate that since a crRNA binding to a virus-specific sequence activates the activity of Cas13a enzymes, will two crRNAs binding to different virus-specific sequences activate the activity of Cas13a enzymes more quickly, making fluorescent signals rise faster?
The experiment also confirmed their hypothesis. When the researchers activated Cas13a enzymes using two different crRNAs, the growth rate of fluorescent signals increased significantly, although the total crRNA levels did not change. By detecting the growth rate of the fluorescent signal rather than the absolute value of the fluorescent signal, they found that using two crRNAs significantly improved the sensitivity of the detection. When one crRNA is used, the sensitivity detected reaches 10,000 virus copies/microliters, while when two different crRNAs are used, the sensitivity is increased to less than 1000 copies/microlith.
To further improve the sensitivity of the test, the researchers developed crRNA testing with three different specific RNA sequences targeting the new coronavirus, which was accurately detected in an experiment that accurately detected five positive samples in five coVID-19 patient nasopharyngeal swabs, and the increase in fluorescent signals was well linear with the number of virus copies in the input sample, further demonstrating that the test could be used to quantify virus levels.
Build an easy signal reading system with your smartphone’s camera.
To demonstrate that this simple detection can be used in environments outside the lab, the researchers built a simple fluorescent signal detection system based on a smartphone camera. To their surprise, the fluorescent signal detection system, based on mobile phone cameras, is an order of magnitude more sensitive than the commercial plate reader used in the lab. Using this simple fluorescent signal detection system, researchers were able to determine in just five minutes that five patients with COVID-19 had a positive nasopharyngeal swab sample.
Fluorescent signal reading system based on smartphone camera design.
Not only are smartphones popular today, but their cameras are highly sensitive, the researchers note in the discussion. What’s more, smartphones usually carry GPS and can be connected to the Internet. This makes it easier to transfer read results to a cloud database and assist contact tracking. Smartphone-based new coronavirus detection systems may play an important role in controlling current and future pandemics.