NEW YORK – Researchers from Pennsylvania State University have harnessed loop-mediated isothermal amplification technology to create a miniaturized COVID-19 test that could be implemented at the point of care.
The test is intended to "solve a gap" between at-home antigen tests, which are fast but not sensitive, and PCR tests, which are the gold standard for sensitivity but can take a day or two to return results, said Weihua Guan, an associate professor at Penn State's school of electrical engineering and computer science.
Guan and his team developed a device with two components: an all-in-one microfluidic cassette and a small handheld analyzer. The single-use disposable cassette is able to automatically heat lyse a saliva sample, while the reusable analyzer contains the electronic, optical, and mechanic components used to detect the presence of SARS-CoV-2. All told, the process takes between 35 and 45 minutes to return a result to a smartphone, depending on the viral load in the sample.
First, someone spits into a conduit connected to a chamber on the cassette, which is then inserted into the analyzer; the sample is then heated for five minutes. Ten microliters of sample are then guided into another section of the cassette via microfluidic channels, dispensed into three separate chambers containing the reagents, and mixed.
Sample preparation for saliva usually requires multistep lysis and purification processes to remove foreign materials from the sample, but Guan's team optimized its microfluidic cartridge to create an extraction-free method of sample prep while reducing the complexity of the device.
In regular benchtop PCR instruments, optical lenses that need a certain focal length are used for detection, but in Guan's device those lenses are eliminated and replaced with optical sensors that are directly attached to the area in the device where photons are emitted from the sample if the virus is present. Eliminating the traditional optical lenses enabled the researchers to shrink the device and make it portable, he said.
The technology used is similar to other RT-LAMP tests for SARS-CoV-2 — of which there have been many — but Guan said the key difference is the elimination of the purification step, thanks to the work the researchers did to optimize saliva sampling. The team tested variable amounts of saliva with its reaction mix to determine the ideal volume that would not destroy the reaction but would have enough virus to be detected. That ideal amount is 10 microliters, he said.
Guan noted that he envisions the test being used more for preliminary screening at a doctor's office or at home, similar to antigen tests, rather than as a confirmatory diagnostic test. LAMP technology, including Guan's device, can be subject to "unexplainable" false positives, so he recommended an additional laboratory PCR test be performed if the initial test was positive.
Because of the potential for false positives, Guan said he and his team are working to introduce more specific signal reporting strategies, such as combining LAMP with CRISPR for higher specificity.
A key problem with antigen tests that many experts have called out is sensitivity, but Guan also noted that sample collection issues can hinder results. At-home antigen tests rely on users who may improperly swab for nasal samples, producing false negative results. But by using saliva, "as long as people can spit a sufficient amount of saliva," the sample can be collected correctly, he said.
Saliva is also less invasive and less subject to variations in the collection process, he added.
The researchers have filed a patent for the device and are currently trying to get more clinical samples to test and compare with PCR results. Earlier this month, the team published a study in ACS Sensors using 120 samples with inactivated SARS-CoV-2 particles spiked into healthy saliva that had 100 percent agreement with PCR, he said. The researchers also tested six archived clinical samples.
"We definitely need to scale up the sample numbers to get a more accurate assessment of the performance," Guan said, adding the team has plans to obtain more clinical samples for further research.
Michael Mahan, a professor at the University of California, Santa Barbara, who is working on an RT-LAMP test for COVID-19 and influenza, said via email that the test is promising but noted that the cost of the device, reagents, and cartridge will determine feasibility. He also said that molecular tests often have two different amplification targets to avoid false negatives from emerging variants, but Guan's test only has one target site as of now.
Guan said that the test is not specific to one variant and because the intended use of the assay is a first-line screening test, the team doesn't intend to change the test to make it variant-specific.
Meantime, Xiaowei Wang, a professor of pharmacology and bioengineering at the University of Illinois Chicago who developed an RT-LAMP method that doesn't require RNA isolation or purification, said via email that the test is a "pretty neat all-in-one system" for COVID-19 testing. He noted that it would be most beneficial at small clinics and community testing centers, since it doesn't require extensive training to operate. However, he agreed that more testing needs to be done to confirm the "robustness of the system," as well as more extensive validation of the instrument via field deployment to collect feedback from customers or users.
As more data is collected, Guan's team is working with Penn State's Center for Medical Innovation to get 510(k) clearance for the technology from the US Food and Drug Administration. After that, Guan said he wants to eventually commercialize the test internationally. While he declined to share what the price would be for the test and device, right now the cartridge costs between $1 and $2 to manufacture and the analyzer costs between $80 and $90. However, mass producing the components would lower the overall cost, he said.
The platform could also conceivably be used for any heat-lysable, saliva-borne diseases, Guan said, such as influenza or herpes.