New Saliva-Based CRISPR Smartphone Assays May Fill Need for Mass and Rapid COVID-19 Testing

One of the few positive things to come out of this deadly global pandemic is how it has accelerated the pace of diagnostic innovation to hyperdrive levels. The latest example of this phenomenon is the rapid development of technology that enables individuals to use their smartphones to find out if they have COVID-19 via analysis of a saliva sample.

The Diagnostic Challenge

• Should we shut down the schools?
• How much progress are we making toward general immunity?
• Should we open the workplace?
• Is it safe for me to leave the home and go to the grocery store?

Mass rapid COVID-19 testing provides the critical data government regulators, scientists, employers and individuals need to make sound decisions about how to keep COVID-19 from spreading.

Molecular real-time reverse transcription polymerase chain reaction (RT PCR) testing represents the current gold standard for COVID-19 testing. But while it is capable of accurate detection, RT PCR test methods do not generate this kind of data. Because tests must be performed at an off-site laboratory using complex RNA extraction methods, individuals who get tested have to wait for days to find out if they are positive or negative. RT PCR tests are also dependent on supplies that are currently hard to get, including nasal swabs that must be inserted into the patient’s nostril to obtain a sample from the sinus cavity, a process that is uncomfortable and, in many cases, must be carried out by a trained health professional.

The good news is that since the public health emergency began, researchers, universities and commercial laboratory companies have made significant progress in developing rapid, accurate and point of care COVID-19 diagnostics. Such progress has been manifested across a number of different fronts.

Home Sample Collection

One of the pathways to mass and rapid COVID-19 testing is the development of products allowing for home collection of test samples. Early in the pandemic, there was understandable skepticism about whether patients are actually capable of swabbing their own nostrils. However, a number of influential studies, including by researchers at the Stanford University School of Medicine published in The Journal of the American Medical Association in June provided critical evidence that patients can, in fact, be trusted to swab themselves.

On April 21, Laboratory Corporation of America’s COVID-19 RT PCR test became the first assay to receive U.S. Food and Drug Administration (FDA) Emergency Use Authorization (EUA) for use on home-collected samples. A month later, Quest Diagnostics received EUA for its own COVID-19 self-collection kit. And as 2020 comes to a close, dozens of other COVID-19 molecular tests have been cleared for home sample collection.

Saliva-Based Testing

A variation on this same theme has been the development of COVID-19 tests that can be performed on saliva samples thereby obviating the need for nasal swab collection altogether. While nobody disputes that saliva testing is cheaper, simpler and easier on the supply chain, there were concerns about its accuracy as compared to testing on samples obtained by nasopharyngeal swabs. But as with home collection, evidence indicating that COVID-19 saliva-based is at least as reliable and accurate as swab-based testing was soon to emerge.

A Yale University study published in The New England Journal of Medicine on Aug. 28 found that the Yale saliva test actually detected the SARS-CoV-2 virus more frequently in patients known to have COVID-19, with 81 percent of the tests coming back positive in the first five days of infections, compared to the 71 percent rate got nasopharyngeal tests. The saliva test also detected more copies of the virus’s genetic material.

A second study from the University of Ottawa published in the Annals of Internal Medicine the very same day was also supportive of saliva testing. The researchers tested nearly 2,000 people who had either mild symptoms of the virus or no symptoms but were at a high risk of infection. Participants collected their own saliva and also underwent the traditional swab test: 34 came back positive in both tests. In 14 cases, the virus was detected in the saliva sample, but not the nasal sample. In 22 cases, the opposite was true.

Even before these studies appeared, the FDA granted its EUA for a COVID-19 for use on saliva samples on April 13. The Rutgers Clinical Genomics Laboratory TaqPath SARS-CoV-2 Assay test is based on the Thermo Fisher Scientific Applied Biosystems TaqPath COVID-19 Combo Kit previously approved for the detection of specific genomic regions of the SARS-CoV-2 nucleocapsid gene, spike gene and ORF1ab region in nasopharyngeal swab, nasopharyngeal aspirate, and bronchoalveolar lavage specimens. But it was modified for use on additional specimen types, including saliva.

Of the roughly 250 COVID-19 assays to gain EUA by year’s end, over a dozen have been approved for use on saliva samples.

Smartphone Testing on Saliva Samples

The next phase in the evolution was the marrying of these simplified sample collection methods with consumer-based, mobile technology allowing for rapid and accurate testing at the point of care. And because just about everybody owns a smartphone, this device would serve as the central node. However, instead of RT PCR, smartphone tests would be built around Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) gene-editing technology.

As with RT PCR, CRISPR diagnostics require the conversion of RNA to DNA, which then must be amplified to improve the accuracy of detection. That would have barred use of CRISPR with smartphones. But researchers were able to clear this hurdle by developing a novel approach allowing CRISPR to be used to detect viral RNA directly, thereby eliminating the need for conversion and amplification.

The California Assay

In a study published in the scientific journal Cell, a team of researchers from Gladstone Institutes, University of California, Berkeley, and University of California, San Francisco outlined the technology for a CRISPR-based test for COVID-19 that uses a smartphone camera to provide accurate results in under 30 minutes.

The researchers built a prototype assay chip that uses the CRISPR/Cas12a enzyme to enhance an amplified viral RNA target’s signal within a saliva sample. They integrated the chip into a smartphone-based fluorescence microscope readout device, which captures and analyzes images to determine whether the virus is present above a threshold concentration. They then tested the technique on 12 people infected with COVID-19 and six healthy controls. Result: The test was just as effective as RT PCR in distinguishing between people with and without the virus. “We believe this smartphone platform, a similar future application, offers the potential to rapidly expand COVID-19 screening capacity, and potentially simplify the verification of contact tracing, to improve local containment and inform regional disease control efforts,” the authors wrote.

The Tulane Assay

A week after the Cell study describing the California 30-minute test, researchers from Tulane University raised the bar by a quarter of an hour when they unveiled their own version of a saliva-based test for COVID-19 that delivers results via a smartphone in 15 minutes. Like the California test, the Tulane assay is based on CRISPR technology capable of detecting SARS-CoV-2 virus RNA directly without the need for conversion and amplification. The researchers say the new saliva-based test is not only faster and more user-friendly but also more sensitive than standard RT PCR tests. “The sensitivity and simplicity of this test, its straightforward sample collection procedure, and the inexpensive nature of the readout device should permit the rapid translation of this approach to COVID-19 testing efforts,” noted one of the Tulane researchers.

Takeaway

 Although neither test has yet received FDA approval, the development of CRISPR-based assays capable of rapid and accurate COVID-19 diagnostics at the point of care via the use of consumer technology is the culmination of innovation across a number of fronts that the pandemic set into motion. The new assays essentially cut out the laboratory middleman making them more efficient and scalable. And if it they truly are as sensitive and reliable as RT PCR tests the way the researchers suggest, they could be a breakthrough in the effort to bring mass and rapid COVID-19 testing to the masses.