The new CRISPR-based test for COVID-19, developed by researchers at the Gladstone Institute, UC Berkeley and UC San Francisco, essentially turns the smartphone camera into a microscope to provide fast and accurate results. Credit: Gladstone Institutions
Imagine swabbing your nostrils, swabbing the device, and getting a readout on your phone in 15 to 30 minutes whether you are infected or not. COVID-19 Virus. This is the vision for a team of scientists at Gladstone Institute, University of California, Berkeley (UC Berkeley), and the University of California, San Francisco (UCSF). And now, they report the scientific progress that brings them closer to making this vision a reality.
One of the main barriers to fighting the COVID-19 epidemic and reopening communities across the country is the availability of a largely rapid test. Knowing who is infected will provide valuable insights for policy makers and citizens about the potential spread and risk of the virus.
Yet, people often have to wait for their results, or even when there is a backlog of laboratory tests. And, the situation is exacerbated by the fact that most infected people have mild or no symptoms, even though they carry and spread the virus.
In a new study published in the scientific journal Cell, Gladstone, UC Berkeley and a team from UCSF have outlined the technology for CRISPR-based testing for COVID-19 that uses a smartphone camera to provide accurate results in less than 30 minutes.
A team of scientists from Gladstone, UC Berkeley and UC San Francisco, including Melanie Tutt (left) and Parinaz Fozoni (right), outlined the technology for rapid, one-step mobile testing that could help fight epidemics and completely reopening communities. . Credit: Gladstone Institutions
“The urgent task is not only to increase testing for the scientific community, but also to provide new testing options,” says Melanie Ott, MD, PhD, director and study leader at the Gladstone Institute of Virology. “The ocean we created could provide fast, low-cost testing to help control the spread of COVID-19.”
The technology was developed in collaboration with Uni Berkeley bioengineer Daniel Fletcher, PhD, as well as Jennifer Dowdna, PhD, a senior investigator at Gladstone, a professor at UC Berkeley, president of the Innovative Genomics Institute, and a Howard researcher. Hughes Medical Institute. Dudna recently won the 2020 Nobel Prize in Chemistry for co-discovery of the CRISPR-CAS genome editing technology that underpins this task.
Not only can their new diagnostic test bring a positive or negative result, it also measures the viral load (or its concentration). SARS-CoV-2, The virus that causes COVID-19 in a given sample).
“When tested frequently, measuring viral load can help determine if the infection is increasing or decreasing,” says Fletcher, who is also a Chen Zuckerberg biohub investigator. “Monitoring a patient’s course of infection can help health care professionals estimate the stage of the infection and predict how much time is needed for recovery, in real time.”
A simple test by direct detection
Current COVID-19 tests use a method called quantitative PCR – the gold standard of testing. However, one issue is that it is necessary to use this technology to test for SARS-Covy-2. DNA. The coronavirus is RNA Virus, which means that in order to use the PCR approach, viral RNA must first be converted into DNA. In addition, the technique is based on a two-step chemical reaction, which involves an amplification step, in which sufficient DNA is provided to make it detectable. Therefore, current tests typically require trained users, specialized reagents, and cumbersome lab equipment, which severely limits where testing can take place and causes delays in achieving results.
As an alternative to PCR, scientists are developing test strategies based on the gene-acquisition technique CRISPR, which is particularly good at identifying genetic material.
All CRISPR diagnostics to date require that viral RNA DNA be detected. Before it can be converted and detected, time and complexity are added. In contrast, the novel approach described in this recent study ignores all conversion and amplification steps to detect viral RNA directly using CRISPR.
“One of the reasons we’re excited about CRISPR-based diagnostics is that there is potential for fast, accurate results based on need,” says Dowdna. “This is especially helpful in places with limited access to testing or when frequent, rapid testing is needed. It can overcome many obstacles with COVID-19. “
Fozouni (left), a graduate student at Otni (right) Lab, is the co-author of the study published in the cell. Credit: Gladstone Institutions
Parinaz Fozouni, a UCSF graduate student working in the TTT lab at Gladstone, has been working on an RNA testing method for HIV for the past few years. But in January 2020, when it became clear that coronavirus was becoming a major issue globally and that testing was a potential hassle, she and her colleagues decided to shift their focus to COVID-19.
“We know that the pit we are developing would be logically appropriate to help in an emergency by allowing for quick testing with minimal resources,” says Eva Fozouni, co-author of the paper with Sungmin’s son and Maria Diaz de Leon Derby. Fletcher’s team at UC Berkeley. “Instead of the well-known CRISPR protein called CAS9, which recognizes and caffeines DNA, we have used CAS13, which captures RNA.”
In the new test, the KS13 protein is attached to the reporter molecule that becomes fluorescent when cut, and then blended into the patient’s sample with a nasal swab. The sample is placed in a device that connects to the smartphone. If the sample contains RNA from SARS-Cavi-2, KS13 will be activated and the reporter will cut the molecule, emitting a fluorescent signal. After that, the smartphone’s camera marrow, essentially converted into a microscope, can detect fluorescence and report that the swab is testing positive for the virus.
“What makes this test really unique is that it uses a one-step response to directly test for viral RNA, just like traditional PCR,” says Evatt, a professor in the Department of Medicine. . As opposed to a two-step process in tests, “says Ot, who is also a professor of medicine at UCSF.” Simple chemistry attached to a smartphone camera reduces investigation time and does not require complex lab devices. Allows quantitative measurement instead. “
Researchers also say that their rocks can be adapted to a variety of mobile phones, making the technology easily accessible.
“We chose to use a mobile phone based on our device because it has an intuitive user interface and a highly sensitive camera that we can use to detect fluorescence,” Fletcher explains. “Mobile phones are also mass-produced and cost-effective, indicating that no specialized laboratory devices are required for this room.”
Accurate and fast results to limit the epidemic
When the scientists tested their device using patient samples, they confirmed that it could very quickly reverse the results for clinically relevant viral load samples. In fact, the device detected a bunch of positive samples in less than 5 minutes. For samples with low viral load, up to 30 minutes are required to separate the device from the negative test.
SAT says, “SARS-Co-Tuna’s latest model Dales suggests that we need to overcome the current epidemic by repeating the test with rapid change. “We hope that with increased testing, we can avoid lockdowns and protect the most vulnerable populations.”
The new CRISPR-based testing not only offers a promising option for quick testing, but using smartphones and avoiding the need for large lab equipment, it is likely to be portable and eventually made available for point-of-care or even home use. And, it can be extended for the diagnosis of other respiratory viruses outside of SARS-COV-2.
In addition, the sensitivity of smartphone cameras, along with their connectivity, GPS and data-processing capabilities, have made them attractive tools for diagnosing diseases in low-resource regions.
“We hope to develop our testing into a device that can upload immediate results to a cloud-based system that maintains patient privacy, which will be important for contact tracing and epidemiological studies,” says Ott. “This type of smartphone-based diagnostic testing could play a crucial role in controlling current and future epidemics.”
References: “Amplification-Free Detection and Mobile Phone Microscopy of SRS-Cavi-2 with CRISPR-KS13A” Knott, Carly Ann. Gray, Michael V. D. Ambrosio, Chunyu Zhao, Neil A. Switz, g. Renuka Kumar, Stephanie I. Stephens, Daniela Bohem, Chia-Lin Tsu, Jeffrey Shu, Abdul Bhuiyan, Max Armstrong, Andrew R. Harris, P-Yi Chen, Jannet m. Ter Sterloh, Anke Meyer-Frank, Bastian Johank, Keith Walcott, Anita Sill, Charles Lengellier, Catherine S. Pollard, Emily D. Crword, Andres S. Pushchnik, Myra Phelps, Amy Kistler, Joseph L. Dercy, Jennifer a. Dudna, Daniel a. Fletcher and Melanie Tatt, 4 December 2020, Cell.
DOI: 10.1016 / j.cell.2020.12.001
Other authors of this study include Gavin J. Knott, Michael V. D’Ambrosio, Abdul Bhuiyan, Max Armstrong and Andrew Harris of UC Berkeley; Carly Ann. Gray, g. Renuka Kumar, Stephanie I. Stephens, Daniela Bohem, Chia-Lin Tsu, Jeffrey Shu, Janet M. Osterloh, Anke Meyer-Frank, and Catherine S. Pollard from Gladstone Institutions; Amy Kistler from Chanyu Zhao, Emily D. Crawford, Andres S. Pushnik, Myra Phelps and Chan Zuckerberg Biohub; Neil A. Switz from San Jose State University; And Charles Langellier and Joseph L. from UCSF. DRC.
The research was supported by the National Institute of Health (NIAID grant 5R61AI140465-03 and NIDA grant 1R61DA048444-01); NIH Rapid Acceleration of the Diagnostics (RADX) program; National Heart, Lung and Blood Institute; The National Institute of Biomedical Imaging and Bioengineering; Department of Health and Human Services (Grant No. 3U54HL143541-02S1); As well as Fast Grants, James b. Through philanthropic support from the Pendleton Charitable Trust, The Rodenberry Berry Foundation and multiple individual donors. This work was also made possible by a generous gift from an anonymous private donor in support of the ANCRR Diagnostics Consortium.
