Three NSF RAPID Grants to Develop Quicker Test for COVID-19 for Holonyak Lab Faculty

Three Nick Holonyak Jr., Micro and Nanotechnology Lab (HMNTL) faculty members have received NSF Rapid Response Research (RAPID) program grants, all of which aim to shorten the amount of time it takes to process a COVID-19 test. Current tests can take as long as five days for results to be returned to the patient. Although more rapid nucleic acid tests that can give a result within an hour have become available, there are reports of a high rate of false negatives among these tests.

With the United States reaching the highest number of SARS-CoV-2 (this particular strain of coronavirus) cases out of any infected country, it is a national imperative to be able to test people before they show symptoms to reduce the spread of the deadly disease.

"As one of the only facilities in the country that incorporates both micro and nanofabrication cleanroom facilities and a BioNanotechnology Laboratory (BNL) under the same roof, HMNTL is proud to meet the moment and provide support to COVID-19 related essential research," said Xiuling Li, HMNTL interim director and Donald Biggar Willett Professor in Engineering.

Here is a more in-depth look at how HMNTL faculty are helping:

Rapid Electrical Detection of COVID-19 at Point-of-Care

A team led by Rashid Bashir, Dean of the Grainger College of Engineering, and Holonyak Lab faculty researcher, has proposed the development of a point-of-care device that uses nasal fluid samples to detect the presence of COVID-19 within 10 minutes.
Current tests are complex and labor-intensive, requiring each sample to be sent to a laboratory for confirmation. The test being developed by Bashir's group will simplify the process by eliminating the need to extract RNA from samples and simplify the test it as a whole. The new test will electrically detect specific nucleic acid molecules associated with the SARS-CoV2-2.

"Our approach can provide for a rapid electrical detection of the RNA amplification using graphene sensors and result in a miniaturized format for the test and also reduce the test's total processing time," said Bashir, Abel Bliss Professor of Engineering, professor of bioengineering, and member of the Center for Genomic Diagnostics.

The team hopes the proposed approach can be expanded beyond COVID-19 detection to become a global health technology that contributes to providing low-cost diagnostics of a number of viruses around the world in a portable and inexpensive way.

Rapid Single-Step Reagentless SARS-CoV-2 Viral Load Test by Detection of Intact Virus Particles

The next COVID-19 detection project combines capturing intact COVID-19 viruses with custom-designed DNA nanostructures so they can be immediately counted with a newly-invented type of biosensor imaging. This process could be completed and produce results in less than 15 minutes.

This new method would allow diagnostic facilities at the point of care to count each virus directly using a new form of ultrasensitive biosensor microscopy that amplifies the magnitude of light scattering produced by the virus when it is illuminated with a laser. To determine if the viewed virus is SARS-CoV-2, customized DNA nanostructure-based capture probes would be immobilized on a photonic crystal biosensor surface. When exposed to a sample, such as material eluted from a nasal swab, the DNA rhombus-shaped "virus net" would selectively attach the virus to the biosensor surface, while allowing all other materials to pass over the sensor without capture.

"Our approach would represent a new paradigm for virus diagnostics that does not require the chemical enzymatic amplification of nucleic acids, and so does not require temperature control, thermal cycles, viral lysis, nucleic acid purification, or fluorescent dyes," said Cunningham, Donald Biggar Willett Professor in Engineering and professor of electrical and computer engineering. "We just capture and count, so it is the simplest possible process, and our sensing method gives a result immediately as the viruses are captured."

The technology used in this method was recently demonstrated as a new form of biosensor microscopy called Photonic Resonator Interference Scattering Microscopy (PRISM), which allows researchers to detect and digitally count virus particles, protein molecules, and a variety of nanoparticles in real time without the use of additional labels or stains.

The team for this research also includes Xing Wang, associate professor of Chemistry, Taylor Canady, postdoc fellow at the Carl R. Woese Institute for Genomic Biology, and Nantao Li, Cunningham's ECE graduate student.

RAPID: Developing a Novel Biosensor for Rapid, Direct, and Selective Detection of COVID-19 using DNA Aptamer-Nanopore 

Holonyak Lab affiliate faculty member Yi Lu is working with Lijun Rong from the University of Illinois at Chicago to develop a biosensor that could detect and differentiate infectious SARS-CoV-2 from the SARS-CoV-2 that have been rendered noninfectious by patient's antibodies or disinfectants. This would allow patients to receive proper treatment in a timely manner, and would allow people who aren't infected or contagious to be released from quarantine.

The project aims to develop a modular and scalable sensor for direct detection of the intact coronavirus using DNA aptamers, short, single-stranded DNA molecules that can selectively bind infectious SARS-CoV-2. When coupled with nanopore, a pore of nanometer size, the result would be able to differentiate the infectious SARS-CoV-2 from the non-infectious forms or other viruses such as flu viruses with a high level of specificity.

"Achieving such a high level of specificity is very important for COVID-19 diagnostics," said Yi Lu, professor of chemistry and bioengineering. "This is because studies have shown that viral RNA levels that are being used in most COVID-19 diagnostic tests do not always correlate with viral transmissibility."

This technique is less resource-intensive than current methods due to not requiring pretreatment or RNA amplification. It also decreases the likelihood of cross contamination. It could also be used to test surface areas to ensure they have been properly sanitized after coming in contact with an infected patient.

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COVID-19 Antigen Test Evaluated by European Scientists

Scientists in Europe recently evaluated the frontline capabilities of a commercially available, 15-minute disposable antigen test to detect COVID-19 infections.

Their findings, reported in Frontiers in Medicine, suggest the test could be useful as part of a broader triage strategy for slowing down the virus, which has infected more than seven million people and caused about 250,000 deaths as of May 4.

“The detection of viral infections in patients attending primary care centres would allow healthcare workers to rapidly identify new outbreak foci and define quarantine measures for high viral shedders and/or suspect patients to limit the spread of the epidemic,” the authors wrote.

The two-phase study examined the sensitivity and specificity of the new test during its development stage in the lab and later on using real-world biological samples from more than 300 previously infected patients.

Overall accuracy was 82 percent in the latter setting, with an overall sensitivity (how often a test correctly generates a positive result) of 57.6 percent and an overall specificity (how often a test correctly generates a negative result) of 99.5 percent.

In other words, the test was able to detect COVID-19 infections in about six out of 10 people, and it was nearly perfect in determining when an infection was not present. The test was more sensitive in patients with higher viral loads, positively identifying an infection in about seven out of 10 people.

The authors say the test — quicker, cheaper and less complicated but not as sensitive as reverse transcription-polymerase chain reaction (RT-PCR) assays, which ID the virus based on its genetic material — could be used to help screen patients during peak periods of the pandemic. Eventually, it could also be especially useful in screening higher-risk populations such as healthcare workers, they said.

The COVID-19 Ag Respi-Strip® test was developed by Belgian company Coris BioConcept, which specializes in rapid diagnostic kits for detecting respiratory and gastrointestinal pathogens like viruses and bacteria.

The test from Coris BioConcept is a type of immunochromatographic assay, or lateral flow test, which detects the presence or absence of a particular substance. Most people may be familiar with another type of lateral flow assay — a pregnancy test.

In the case of the COVID-19 Ag Respi-Strip, the antigen test uses a sample from a nasopharyngeal swab, which looks like a long, flexible Q-tip that enters through one nostril and extends down the nasal passage close to a person’s outer ear.

An antigen test works by looking for proteins on the surface of the virus. Coris BioConcept partly based the test on previous virology research on SARS-CoV-1, which caused the 2002-03 SARS epidemic. In fact, the two are so similar that the COVID-19 Ag Respi-Strip cannot differentiate between SARS-CoV-1 and -2.

The authors estimate the 15-minute antigen test, which can be conducted at point-of-care facilities following a few user-friendly protocols, could reduce the number of laboratory tests using RT-PCR by more than 13 percent.

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Quidel Receives FDA Emergency Use Authorization for Rapid Antigen COVID-19 Diagnostic Assay

Quidel Corporation, a provider of rapid diagnostic testing solutions, cellular-based virology assays and molecular diagnostic systems, announced today that Quidel has received Emergency Use Authorization (EUA) from the U.S. Food and Drug Administration (FDA) to market its Sofia® 2 SARS Antigen FIA, a rapid point-of-care test to be used with the Sofia 2 Fluorescent Immunoassay Analyzer for the rapid detection of SARS-CoV-2 in nasal or nasopharyngeal specimens from patients meeting the Centers for Disease Control and Prevention’s (CDC) criteria for suspected COVID-19 infection.

Sofia 2 is Quidel’s next-generation version of its best-selling Sofia instrumented system. Sofia 2 utilizes the original Sofia fluorescent chemistry design while improving upon the graphical user interface and optics system to provide an accurate, objective and automated result in 15 minutes. The next-generation Sofia 2 system also comes connected to Virena®, Quidel’s data management system, which provides aggregated, de-identified testing data in near real-time.

The Sofia 2 instrument also offers 2 distinct workflows: depending upon the user’s choice, the Sofia 2 SARS Antigen FIA cartridge is placed inside Sofia 2 for automatically timed development (WALK AWAY Mode); or test cartridges can be placed on the counter or bench top for a manually timed development and then placed into Sofia 2 to be scanned (READ NOW Mode), allowing the user to markedly increase testing throughput per hour.

“In the fight against COVID-19, our employees are truly making a difference, and I am tremendously proud of our organization’s ability to quickly develop and mobilize an accurate rapid antigen test,” said Douglas Bryant, president and chief executive officer of Quidel Corporation. “The EUA for our Sofia 2 SARS Antigen FIA allows us to arm our healthcare workers and first responders with a frontline solution for COVID-19 diagnosis, accelerating the time to diagnosis and potential treatment of COVID-19 for the patient.”

The assay is currently available for sale in the United States under EUA, and Quidel is now shipping the product to its customers. Quidel offers several other Sofia assays for sale, which are FDA cleared and CLIA waived, including tests for Influenza A and B, Respiratory Syncytial Virus (RSV), Group A Strep, and a 12-minute finger-stick whole blood test for Lyme Disease. In addition, Quidel also markets Sofia® tests for Lyme Disease, Legionella and S. pneumoniae in Europe.

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CRISPR-based Technology Spots COVID-19

Researchers have developed a new technology that flexibly scales up CRISPR-based molecular diagnostics, using microfluidics chips that can run thousands of tests simultaneously. A single chip’s capacity ranges from detecting a single type of virus in more than 1,000 samples at a time to searching a small number of samples for more than 160 different viruses, including the COVID-19 virus.

Called Combinatorial Arrayed Reactions for Multiplexed Evaluation of Nucleic acids (CARMEN), this technology — validated on patient samples — provides same-day results and could someday be harnessed for broad public-health efforts.

The work appears in Nature, led by co-first authors Cheri Ackerman and Cameron Myhrvold, both postdoctoral fellows at the Broad Institute of MIT and Harvard. Paul Blainey, core member of the Broad Institute and associate professor in the Department of Biological Engineering at MIT, and Pardis Sabeti, institute member at Broad, professor at Harvard University, and Howard Hughes Medical Institute Investigator, are co-senior authors.

“The current pandemic has only underscored that rapid and sensitive tools are critical for diagnosing, surveilling, and characterizing an infection within a population,” said Sabeti. “The need for innovative diagnostics that can be applied broadly in communities has never been more urgent.”

“CRISPR-based diagnostics are an attractive tool for their programmability, sensitivity, and ease of use,” said Myhrvold. “Now, with a way to scale up these diagnostics, we can explore their potential for comprehensive approaches — for example, enabling clinicians to see if patients are harboring multiple infections, to rule out a whole panel of diseases very quickly, or to test a large population of patients for a serious infection.”

Miniaturizing CRISPR diagnostics

To build a testing platform with this capacity, the team turned to microfluidics, adapting and improving on technology developed in 2018 by Blainey’s lab. The researchers created rubber chips, slightly larger than a smartphone, with tens of thousands of “microwells” — small compartments designed to each hold a pair of nanoliter-sized droplets. One droplet contains viral genetic material from a sample, and the other contains virus-detection reagents.

“The microwell chips are made like a stamp — it’s rubber poured over a mold,” explained Ackerman. “We’re easily able to replicate and share this technology with collaborators.”

The detection approach used on the chips is adapted from the CRISPR-based diagnostic SHERLOCK, first described in 2017 and developed by team of scientists from the Broad Institute, the McGovern Institute for Brain Research at MIT, the Institute for Medical Engineering & Science at MIT, and the Wyss Institute for Biologically Inspired Engineering at Harvard University.

To use the CARMEN platform, researchers first extract viral RNA from samples and make copies of this genetic material, similar to the preparation process for RT-qPCR diagnostics currently used for suspected COVID-19 cases. The researchers then add a unique fluorescent color dye to each prepared sample and divide the mixture into tiny droplets.

The detection mixtures, on the other hand, contain the CRISPR protein Cas13, a guide RNA that looks for a specific viral sequence, and molecules to report the results. These mixtures are also color-coded and separated into droplets.

Thousands of droplets from the samples and detection mixtures are then pooled together and loaded onto a chip in a single pipetting step. Each microwell in the chip catches two droplets. When a detection droplet finds its target — a specific viral genetic sequence — in a sample droplet in the same microwell, a signal is produced and detected by a fluorescence microscope. The entire protocol, from RNA extraction to results, takes under eight hours.

“Uniting these two technologies in a single platform gives us exciting new capabilities to investigate clinical and epidemiological questions,” said co-author Gowtham Thakku, an MIT graduate student in Broad’s Infectious Disease and Microbiome Program.

CARMEN enables more than 4,500 tests on a single microfluidics chip, which can apply to patient samples in a variety of ways using the available fluorescent codes. For example, a single chip could simultaneously test 1,048 samples for a single virus, or five samples for 169 viruses. The capacity can be easily scaled up further by adding more chips: “We normally run four or five chips in a single day,” noted Ackerman.

Multiplexing capabilities

To showcase the platform’s multidiagnostic capabilities, the team developed a strategy for rapidly testing dozens of samples for the 169 human-associated viruses that have more than 10 published genome sequences. The researchers tested this detection panel against 58 patient samples, using multiple chips. They additionally applied CARMEN on patient samples to differentiate between subtypes of influenza A strains and to detect drug-resistance mutations in HIV.

The team also incorporated detection mixtures for SARS-CoV-2 — the virus that causes COVID-19 — and other respiratory pathogens to demonstrate, using synthetic viral sequences, how the assay can be rapidly adapted to detect emerging viruses.

“CARMEN offers both impressive throughput and flexibility in diagnostic testing,” said co-author Catherine Freije, a Harvard graduate student in the Sabeti lab.

The researchers report that the platform’s sensitivity is comparable to previously published SHERLOCK assays, and they are continuing to improve and validate CARMEN using additional clinical samples. Coupled with the successful testing data from patient samples described in Nature today, this approach could be readily translatable in the clinic, according to the team.

“This miniaturized approach to diagnostics is resource-efficient and easy to implement,” said Blainey. “New tools require creativity and innovation, and with these advances in chemistry and microfluidics, we’re enthusiastic about the potential for CARMEN as the community works to beat back both COVID-19 and future infectious disease threats.”

Support for this study was provided in part by Howard Hughes Medical Institute, the Koch Institute for Integrative Cancer Research Bridge Project, an MIT Deshpande Center Innovation Award, the Merkin Institute for Transformative Technologies in Healthcare, a Burroughs Wellcome Fund CASI Award, the Defense Advanced Research Projects Agency (DARPA) grant D18AC00006, and the NIH (F32CA236425).

Source: The Havard Gazette

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Mammoth Biosciences Announces Peer-Reviewed Validation Of Its Rapid, CRISPR-Based COVID-19 Diagnostic

Mammoth Biosciences announced the publication of a study demonstrating the power of its platform to detect SARS-CoV-2 from respiratory swab RNA extracts in under 45 minutes. The study, published in Nature Biotechnology, contains the first peer-reviewed data using CRISPR diagnostics for COVID-19, with the largest set of patient samples to-date.

There is an urgent need for rapid and accessible testing of the novel coronavirus for an effective public health response. The current and most common method for diagnosing SARS-CoV-2 is through quantitative polymerase chain reaction (qRT-PCR), which is restricted for use within specialized laboratories. As a result, the typical turnaround time for screening and diagnosing patients with suspected SARS-CoV-2 has been more than 24 hours, a pace that is far too slow to keep up with such a contagious disease.

Mammoth Biosciences has harnessed the power of CRISPR to offer a faster, lower-cost and visual alternative to traditional qRT-PCR assays. The company’s CRISPR-based diagnostic assay, DETECTR™, can deliver results in under 45 minutes as visualized on a lateral flow strip, similar to an at-home pregnancy test. DETECTR does not require a complex laboratory setting; it can be performed with portable heat blocks and readily available, “off-the-shelf” reagents and disposable lateral flow strips. The assay offers similar levels of sensitivity and specificity to qRT-PCR tests, with 95 percent positive predictive agreement and 100 percent negative predictive agreement.

The study was led by infectious disease expert Dr. Charles Chiu, researchers from the Department of Laboratory Medicine at the University of California, San Francisco (UCSF), along with Mammoth’s Chief Technology Officer Dr. Janice Chen and Research Lead Dr. James Broughton, and the California Department of Public Health. The researchers validated the method using contrived reference samples and clinical samples from US patients, including 36 patients with COVID-19 infection and 42 patients with other viral respiratory infections.

“We need faster, more accessible and scalable diagnostics. The point-of-care testing space is ripe for disruption and CRISPR diagnostics have the potential to bring reliable testing to the most vulnerable environments.,” says Mammoth’s Chief Technology Officer Janice Chen. “Because CRISPR can be programmed to detect any DNA or RNA sequence, we have been able to reconfigure our DETECTR platform within days to detect the SARS-CoV-2 virus from one of the first confirmed cases in the U.S., made possible by our collaboration with Dr. Charles Chiu at UCSF.”

From its inception, Mammoth Biosciences has focused on leveraging the diagnostic capabilities of CRISPR to develop decentralized, point-of-care tests for a variety of diseases. Shortly after the outbreak of SARS-CoV-2 in Wuhan, China, Mammoth began building its COVID-19 diagnostic and validated the efficacy of its CRISPR-based protocols on patient samples in less than two weeks. In mid-February, Mammoth published its white paper, open-sourcing its CRISPR-based detection protocols, and in early March, published the preprint containing initial patient sample validation.

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