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Imagine cleaning your nostrils, putting the swab in a device, and getting a reading on your phone in 15 to 30 minutes that tells you if you are infected with the COVID-19 virus. This has been the vision of a team of scientists from the Gladstone Institutes, the University of California, Berkeley (UC Berkeley) and the University of California, San Francisco (UCSF). And now, they report a scientific breakthrough that brings them closer to making this vision a reality.
One of the main obstacles to fighting the COVID-19 pandemic and fully reopening communities across the country is the availability of massive rapid tests. Knowing who is infected would provide valuable information about the possible spread and threat of the virus for both policy makers and citizens.
However, people often have to wait several days for results, or even longer when there is a delay in the processing of lab tests. And the situation is made worse by the fact that most infected people have mild or no symptoms but still carry and spread the virus.
In a new study published in the scientific journal Cell, the team from Gladstone, UC Berkeley and UCSF has outlined the technology for a CRISPR-based test for COVID-19 that uses a smartphone camera to provide accurate results in less than 30 minutes.
“It has been an urgent task for the scientific community not only to increase testing, but also to provide new testing options,” says Melanie Ott, MD, PhD, director of the Gladstone Institute of Virology and one of the study leaders. “The assay we developed could provide low-cost, rapid tests to help control the spread of COVID-19.”
The technique was designed in collaboration with UC Berkeley bioengineer Daniel Fletcher, PhD, as well as Jennifer Doudna, PhD, who is a principal investigator at Gladstone, a professor at UC Berkeley, president of the Innovative Genomics Institute, and a researcher at the Howard Hughes Medical Institute. Doudna recently won the 2020 Nobel Prize in Chemistry for co-discovering CRISPR-Cas genome editing, the technology behind this work.
Your new diagnostic test can not only produce a positive or negative result, but it also measures the viral load (or the concentration of SARS-CoV-2, the virus that causes COVID-19) in a given sample.
“When combined with repeat testing, measuring viral load could help determine whether an infection is increasing or decreasing,” says Fletcher, who is also a researcher at Chan Zuckerberg Biohub. “Tracking the course of a patient’s infection could help healthcare professionals estimate the stage of infection and predict, in real time, how long it is likely to take for recovery.”
Simpler testing through direct detection
Current COVID-19 tests use a method called quantitative PCR, the gold standard of testing. However, one of the problems with using this technique to test for SARS-CoV-2 is that it requires DNA. The coronavirus is an RNA virus, which means that to use the PCR approach, the viral RNA must first be converted to DNA. Furthermore, this technique relies on a two-step chemical reaction, including an amplification step to provide enough DNA to be detectable. Therefore, today’s tests generally require skilled users, specialized reagents, and cumbersome laboratory equipment, greatly limiting where tests can be performed and causing delays in receiving results.
As an alternative to PCR, scientists are developing testing strategies based on CRISPR gene editing technology, which excels at the specific identification of genetic material.
All CRISPR diagnostics to date have required viral RNA to be converted to DNA and amplified before it can be detected, adding time and complexity. In contrast, the novel approach described in this recent study omits all conversion and amplification steps, using CRISPR to directly detect viral RNA.
“One of the reasons we’re excited about CRISPR-based diagnostics is the ability to get fast and accurate results at the point of need,” says Doudna. “This is especially useful in places with limited access to testing, or when rapid and frequent testing is needed. It could eliminate many of the bottlenecks that we have seen with COVID-19.”
Parinaz Fozouni, a UCSF graduate student working in Ott’s Gladstone lab, had been working on an RNA detection system for HIV for the past several years. But in January 2020, when it became clear that the coronavirus was becoming a bigger problem globally and that the tests were a potential danger, she and her colleagues decided to shift their focus to COVID-19.
“We knew that the trial we were developing would be a logical fit to help the crisis by allowing rapid tests with minimal resources,” says Fozouni, who is a co-author of the article, along with Sungmin Son and María Díaz de León Derby from El Equipo de Fletcher. at UC Berkeley. “Instead of the well-known CRISPR protein called Cas9, which recognizes and cleaves DNA, we used Cas13, which cleaves RNA.”
In the new test, the Cas13 protein is combined with a reporter molecule that turns fluorescent when cut and then mixed with a patient sample from a nasal swab. The sample is placed in a device that connects to a smartphone. If the sample contains SARS-CoV-2 RNA, Cas13 will become activated and cut the reporter molecule, causing the emission of a fluorescent signal. Then the smartphone camera, essentially turned into a microscope, can detect the fluorescence and report that a swab tested positive for the virus.
“What really makes this test unique is that it uses a one-step reaction to directly test viral RNA, as opposed to the two-step process in traditional PCR tests,” says Ott, who is also a professor at the Department of Medicine. at UCSF. “The simpler chemistry, combined with the smartphone camera, reduces detection time and does not require complex laboratory equipment. It also allows the test to return quantitative measurements rather than simply a positive or negative result.”
The researchers also say their trial could be adapted to a variety of mobile phones, making the technology easily accessible.
“We chose to use mobile phones as the basis for our detection device, as they have intuitive user interfaces and highly sensitive cameras that we can use to detect fluorescence,” explains Fletcher. “Mobile phones are also mass-produced and cost-effective, showing that specialized laboratory instruments are not necessary for this trial.”
Fast and accurate results to limit the pandemic
When the scientists tested their device on patient samples, they confirmed that it could provide a very fast turnaround time of results for samples with clinically relevant viral loads. In fact, the device accurately detected a set of positive samples in less than 5 minutes. For samples with low viral load, the device required up to 30 minutes to distinguish it from a negative test.
“Recent SARS-CoV-2 models suggest that frequent testing with a fast response time is what we need to overcome the current pandemic,” says Ott. “We hope that with increased testing, we can avoid lockdowns and protect the most vulnerable populations.”
Not only does the new CRISPR-based test offer a promising option for rapid testing, but by using a smartphone and avoiding the need for bulky lab equipment, it has the potential to become portable and eventually be available to the point of care or even for home use. And it could also be expanded to diagnose other respiratory viruses in addition to SARS-CoV-2.
Additionally, the high sensitivity of smartphone cameras, along with their connectivity, GPS, and data processing capabilities, have made them attractive tools for diagnosing disease in low-resource regions.
“We hope to develop our test on a device that can instantly upload results to cloud-based systems while maintaining patient privacy, which would be important for contact tracing and epidemiological studies,” says Ott. “This type of smartphone-based diagnostic test could play a crucial role in controlling current and future pandemics.”
Reference: Fozouni P, Son S, Díaz de León Derby M, et al. Detection without amplification of SARS-CoV-2 with CRISPR-Cas13a and mobile phone microscopy. Cell. 2020: S0092867420316238. doi: 10.1016 / j.cell.2020.12.001
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