NEW YORK – Uppsala University spinout Readily Diagnostics is using technology built off 30 years of research as the basis of a point-of-care, instrument-free test that would detect and differentiate between multiple respiratory diseases.
The Swedish firm is using padlock probes, which involve a DNA molecule hybridizing and finding a target in a sample, but has added a proprietary twist to speed up the reaction time, Readily Diagnostics CEO Liza Löf said in an interview. The probes were first invented in the 1990s by Ulf Landegren and Mats Nilsson and have been used in other companies' technology, but while working in Landegren's laboratory, Löf developed a "secret sauce" to facilitate more rapid diagnosis.
Fellow Uppsala University spinout Rarity Bioscience is also using a combination of padlock probes and rolling circle amplification, a method of isothermal amplification, in its SuperRCA cell-free and genomic DNA detection technology and has plans to tap into the clinical oncology testing market, in addition to its targeted mutation assay kits for oncology research applications.
With traditional padlock probes, the probes bind to the target and are then closed in by enzymatic ligation. This forms a circle that is then amplified by rolling circle amplification, creating a long DNA molecule that collapses into a bundle that can be detected via fluorescence or nanoparticles, Löf said. Any free padlocks that are not bound to a target are then cleaned and removed from the reaction sequentially. The successful binding of the padlock probe means the sample contained virus particles, she noted.
But Löf's innovation came with the invention of a "cleanup [oligonucleotide]," called the Homogenous Smartlock RCA, that is present in the same reaction when the rolling circle amplification begins and binds to the free padlocks and keeps them in the reaction. Because they are present in the same reaction following amplification, they can re-bind to more targets and generate a new rolling circle amplification.
"Since we don't have to remove the free padlocks, everything can occur in one tube in one reaction at the same time," Löf said. "Upon ligation, this amplification occurs … simultaneously … and that makes the whole thing happen really, really fast."
The test the company is developing relies on the patented Smartlock RCA and the padlock probes to detect respiratory viruses in 15 to 30 minutes from a nasal swab sample. It can use either nanoparticle detection via beads built into a microfluidic chip that cause agglutination that can be seen by the naked eye, or fluorophores read via flow cytometry or an ELISA reader, she said.
The test will detect three types of influenza A, one type of influenza B, respiratory syncytial virus, and SARS-CoV-2.
The padlock probes can be directed against different parts of the genome depending on what virus is being detected and can differentiate between different variants, according to Löf.
In preliminary data that the company expects to publish this year, the respiratory virus test for influenza A/B, respiratory syncytial virus, and SARS-CoV-2 showed 95 percent sensitivity and specificity between 95 and 100 percent.
The company is developing its technology for use in multiple ways, including as a point-of-care microfluidic chip-based assay that would be used for clinical applications as well as flow cytometry- and ELISA-based quantitative tests for research use only. The company intends to seek European and US Food and Drug Administration approval for the point-of-care version, Löf said, and hopes to receive certification under Europe's In Vitro Diagnostic Regulation by 2027 for use at urgent care facilities or home testing under a doctor's telehealth supervision.
The research version of the test could be available later this year and may be used for more rapid population screening, Löf said. Many research laboratories that are collecting large volumes of samples "are hit by very high costs" as they attempt to distinguish between different types of viruses using multiplex PCR and sequencing, and a faster method would be useful to those labs, she added.
Research labs are "drowning in the high price of PCR analysis and sequencing analysis," Löf said, and the company has thus far received a lot of interest in the research-use version of its assay.
The technology is currently being validated with clinicians at Uppsala University using samples that have already been evaluated with qPCR, and the firm is collaborating with research groups in Northern Europe to screen their biobanks. Later this year, Readily Diagnostics plans to set up production for the research-use-only product, she added.
In addition to respiratory viruses, Readily Diagnostics is also developing assays for tuberculosis, sexually transmitted infections, and hepatitis C, Löf said. The only thing that needs to change to adapt the test to another disease is the actual probe, Löf noted. Antibiotic susceptibility testing is another application for the technology, as long as the mutation causing resistance is known.
"As long as we know the type of mutations that we're looking for, we can detect them," Löf said.