One-step rapid mobile test could help fight pandemic and completely reopen communities – ScienceDaily

One of the main obstacles to tackling the COVID-19 pandemic and fully reopening communities across the country is the availability of rapid mass testing. Knowing who is infected would provide valuable information about the potential spread and threat of the virus for policy makers and citizens.

Yet people often have to wait several days for their results, or longer if there is a delay in processing lab tests. And the situation is made worse by the fact that most of those infected have mild or no symptoms, while still carrying and spreading the virus.

In a new study published in the scientific journal Cell, the Gladstone, UC Berkeley and UCSF team described the technology for a CRISPR 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 chiefs leader of the study. “The test we have developed could provide quick and inexpensive 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 senior researcher at Gladstone, professor at UC Berkeley, president of Innovative Genomics. Institute and Howard Hughes Medical Institute researcher. Doudna recently won the 2020 Nobel Prize in Chemistry for co-discovering genome editing of CRISPR-Cas, the technology behind this work.

Not only can their new diagnostic test produce a positive or negative result, 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 repeated testing, measuring viral load could help determine whether an infection is increasing or decreasing,” says Fletcher, who is also a Chan Zuckerberg Biohub researcher. “Monitoring the progress 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 to heal.

Easier testing thanks to 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 in order to use the PCR approach, the viral RNA must first be converted into DNA. In addition, this technique relies on a two-step chemical reaction, including an amplification step to provide enough DNA to make it detectable. Thus, current testing typically requires trained users, specialized reagents, and cumbersome laboratory equipment, which significantly limits where testing can take place and causes delays in receiving results.

As an alternative to PCR, scientists are developing testing strategies based on CRISPR gene editing technology, which excels in 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, which adds time and complexity. In contrast, the new approach described in this recent study skips all the 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 particularly useful in places where access to testing is limited, or where rapid and frequent testing is required. This 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 a few years. But in January 2020, when it became clear that the coronavirus was becoming a bigger problem in the world and testing was a potential trap, she and her colleagues decided to focus on COVID-19.

“We knew that the test we were developing would be a logical solution to help the crisis by enabling rapid testing with minimal resources,” says Fozouni, co-first author of the article, with Sungmin Son and María Díaz de León Fletcher’s Team Derby 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 fluoresces 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 be activated and cut the reporter molecule, causing a fluorescent signal to be emitted. Then the smartphone’s camera, essentially converted to a microscope, can detect fluorescence and signal that a swab has tested positive for the virus.

“What really makes this test unique is that it uses a one-step reaction to directly test for viral RNA, as opposed to the two-step process in traditional PCR tests,” says Ott, who is also a professor. in the Department of Medicine. at UCSF. “The simpler chemistry, combined with the smartphone camera, reduces detection time and does not require complex lab equipment. It also allows the test to produce quantitative measurements rather than just a positive or negative result.”

The researchers also say their test could be adapted for a variety of mobile phones, making the technology easily accessible.

“We chose to use mobile phones as the basis of our detection device because they have intuitive user interfaces and very sensitive cameras that we can use to detect fluorescence,” says Fletcher. “Cell phones are also mass produced and cost effective, demonstrating that specialized laboratory instruments are not required for this test.”

Accurate and rapid results to limit the pandemic

When the scientists tested their device using samples from patients, they confirmed that it could provide very fast turnaround 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 a low viral load, the device took up to 30 minutes to distinguish it from a negative test.

“Recent models of SARS-CoV-2 suggest that frequent testing with a rapid turnaround time is what we need to overcome the current pandemic,” says Ott. “We hope that with an increase in 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 possibly be available for the point. on duty or even at home. And, it could also be extended to diagnose other respiratory viruses beyond SARS-CoV-2.

In addition, 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 areas.

“We hope to develop our test into a device capable of instantly uploading 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.”

About the research project

The study titled “Detection without amplification of SARS-CoV-2 with CRISPR-Cas13a and mobile phone microscopy” has been published online by Cell December 4, 2020.

Other authors of the study include Gavin J. Knott, Michael V. D’Ambrosio, Abdul Bhuiya, Max Armstrong and Andrew Harris of UC Berkeley; Carley N. Gray, G. Renuka Kumar, Stephanie I. Stephens, Daniela Boehm, Chia-Lin Tsou, Jeffrey Shu, Jeannette M. Osterloh, Anke Meyer-Franke and Katherine S. Pollard of Gladstone Institutes; Chunyu Zhao, Emily D. Crawford, Andreas S. Puschnick, Maira Phelps and Amy Kistler of Biohub Chan Zuckerberg; Neil A. Switz of San Jose State University; and Charles Langelier and Joseph L. DeRisi of UCSF.

The research was supported by the National Institutes of Health (NIAID grant 5R61AI140465-03 and NIDA grant 1R61DA048444-01); the NIH Rapid Acceleration of Diagnostics (RADx) program; the National Institute of Heart, Lungs and Blood; the National Institute of Biomedical Imaging and Bioengineering; the Ministry of Health and Social Services (grant n ° 3U54HL143541-02S1); as well as through philanthropic support from the Fast Grants, the James B. Pendleton Charitable Trust, the Roddenberry Foundation and several individual donors. This work was also made possible thanks to a generous donation from an anonymous private donor in support of the ANCeR diagnostic consortium.