The Path to At-Home PCR-based COVID Diagnostics

“The last barrier in molecular testing, even beyond COVID, has been at-home testing with high accuracy—a way to encourage patients to seek testing because it affords them a sense of privacy and control. At-home testing also minimizes public health risks for infectious diseases, because it enables earlier isolation. The pandemic highlighted the need for at-home tests and I don’t see that trend going away,” said Debkishore Mitra, Ph.D., Chief Technology Office and Co-Founder of Lucira, one of three companies that makes at-home PCR-based diagnostics for COVID.

Before COVID, most PCR diagnostic samples were analyzed on large PCR machines in certified central reference labs (such as Quest or LabCorp) or certified clinical hospital labs by highly trained personnel. Now, however, there are 14 at-home or point of care (POC) tests (see Table), out of 278 molecular diagnostics for SARS-CoV-2, that have received emergency use approvals (EUAs) by the FDA as of 6/10/22, the last time their website was updated.

At-home and POC PCR-based COVID tests provide molecular diagnostics close to the sensitivity and accuracy of the lab-based, gold-standard, RT-PCR tests. But the footprint of the analyzer is smaller, they are simpler to use, and their sample-to-answer speed is under an hour rather than days, more similar to the rapid antigen tests. However, PCR-based tests, even POC and at-home, can detect infection earlier than rapid antigen tests can because of their higher sensitivity.

At-home rapid isothermal PCR/NAAT tests

Lucira, Cue, and Detect make the three at-home PCR-based tests. Manufacturing and marketing of their products are still ramping-up so they are not as widely available or known as the rapid antigen tests. According to Mitra, “Becoming available in pharmacies is something we are working toward, but during the last COVID surge, we were able to provide tests really quickly to a wide range of customers via Amazon or our website.”

At-home PCR-based COVID tests come with a small, palm-sized base where either a tube or cartridge containing a nasal swab sample is placed, but for Cue and Detect, the base is reusable and higher in cost (a few hundred dollars) than Lucira’s single-use base. A spokesperson from Cue wrote in an email that among Cue’s customers are universities, Google, and sports associations such as the Major League Baseball.

Isothermal PCR v. RT-PCR

At-home rapid PCR tests are nucleic acid amplification tests (NAAT) that rely on loop-mediated amplification (LAMP) technology, a special type of isothermal NAAT. Isothermal amplification doesn’t cycle between different temperatures as is required for RT-PCR. Colloquially NAAT applications are called ‘PCR-based’ because they involve amplification but NAAT is a more accurate term that covers both isothermal amplification and RT-PCR-based thermocycling amplification. At-home tests are only possible because they are isothermal NAATs. It hasn’t yet been possible to achieve the thermocycling necessary for RT-PCR, down to the size of a palm-sized base analyzer.

Most isothermal NAATs get close to the accuracy and sensitivity of RT-PCR. All are still at least 10-fold more sensitive than antigen-based tests—so they are able to detect infection pre-symptomatically.

Mitra explained that Lucira was able to pivot quickly to COVID because they were close to launching an at-home isothermal NAAT diagnostic for influenza right before the pandemic. They had already developed colorimetric chemistry so amplification steps could be performed at a single temperature and monitored with cheap photo sensors rather than fluorescent probes that require more product and therefore more amplification cycles. And Lucira used microfluidics to minimize user steps. Similarly, Cue also performs chemistry in cartridges and electrochemistry for the detection step.

POC rapid PCR tests

Now, several POC options exist for NAAT-based COVID diagnosis (either isothermal or RT-PCR-based) where the amplification equipment ranges in size from a coffee maker to a microwave. All have simplified instrumentation so user steps are minimal, which allows them to be used without highly trained personnel. However, they still are not small, affordable, or simple enough for at-home use.

Thermo Fisher Scientific’s Accula was one of the early POC RT-PCR machines. According to the company’s website, “The Accula SARS-CoV-2 Test accelerates PCR virus detection using our proprietary Oscillating Amplification Reaction (Oscar) PCR technology that enables shortened thermocycling times and faster PCR completion without the need for costly thermal cycler hardware and optical detection systems used in laboratory-based qPCR.”

Even before the pandemic, Cepheid and BioFire were making RT-PCR machines for molecular diagnostics that did not need to be in a reference lab. However, their products are considered ‘near patient’ rather than fully POC because they still require trained personnel and are larger machines so can’t be used in a CLIA-waived setting. They are more likely to exist in hospital reference labs.

DASH: Rapid RT-PCR in a box

The smallest RT-PCR POC option received its EUA in March of 2022 and is called DASH: Diagnostic Analyzer for Selective Hybridization—created by Minute Molecular Diagnostics. Chad Achenbach, M.D., Professor of Medicine, Northwestern University, who independently verified and published the accuracy and sensitivity of the technology, refers to DASH as ‘PCR in a Box’.

“Innovative engineering and fabrication technology enabled thermocycling in a small machine. The company also relied on microfluidics and software integration so DASH would be easy to use.” Achenbach said Minute Molecular Diagnostics (M2Dx) first developed the technology for diagnosing tuberculosis and other infectious diseases more common in low-resourced countries, which enabled the company to switch gears quickly when COVID arrived. The company is working on making the product commercially available, according to Achenbach.

Saliva-based samples for PCR COVID Dx

Using saliva for COVID diagnostics is easier for the patient and logistically, thereby increasing access to testing, especially in low-resourced areas. Detection of SARS-CoV-2 in saliva has been shown in several studies to be just as sensitive, if not more, especially for omicron, than from nasopharyngeal samples, explained Anne Wyllie, Ph.D., Research Scientist, Yale School of Public Health. Early in the pandemic, Wyllie pioneered a direct-saliva protocol for SARS-CoV-2—obviating the need for RNA extraction. Her group’s protocol is open source and available on Salivadirect.org. “Most COVID tests were initially validated on swab-based nasal samples because that was the most common sample type for respiratory diseases,” said Wyllie. But now more than 180 reference labs have been CLIA-certified to test for SARS-CoV-2 on saliva samples, she added. MicroGem has the first and so far, only EUA for POC testing on saliva but Wyllie can attest, based on the number of companies that have accessed their protocol, that many more are in the works.