TS Digest Issue | July 2023 | Elusive Tsetse Pheromones

Diagnostic platforms for point-of-care settings must be sensitive, easy, and fast. In a paper recently published in ACS Central Science, researchers showcased just that: a new diagnostic platform that rapidly detects viral RNA with a simple bioluminescent readout.¹

Scientists commonly identify pathogens by their nucleic acid fingerprints. Quantitative polymerase chain reaction (qPCR) is the gold-standard method, but diagnostics developers are adopting a quicker and easier alternative: recombinase polymerase amplification (RPA), which amplifies samples at 37°C in 20 minutes.^2,3 “RPA is a fantastic technique famous for amplifying DNA super, super quickly,” said Helena de Puig, a biomedical engineer at the Massachusetts Institute for Technology, who was not involved in the study.

The beauty of bioluminescence is its simplicity. 

—Maarten Merkx, Eindhoven University of Technology

To detect RNA, Maarten Merkx, a biomedical engineer at Eindhoven University of Technology and author of the study, along with his team, combined reverse transcription with RPA (RT-RPA) to rapidly transcribe viral RNA and amplify the resulting double stranded DNA (dsDNA). 

The lower temperature in RPA increases the risk of nonspecific amplification, so Merkx sought a sensitive solution. Recent successful COVID-19 diagnostics used CRISPR enzymes for specificity.² While those methods leveraged Cas12a and Cas13a nucleases, which trigger collateral cleavage of nucleic acids along with target sequence cleavage, Merkx had a different approach in mind. He chose endonuclease dead Cas9 (dCas9), which finds specific sequences in the dsDNA but lacks the machinery to cleave them, for his assay. 

For the detection part, the team borrowed from nature.

“The beauty of bioluminescence is its simplicity,” said Merkx. The team split luciferase, an enzyme that produces bioluminescence, between two dCas9 proteins. When the dCas9 proteins bound to neighboring target sequences, the luciferase fragments combined and enabled bioluminescence, which the researchers captured with a digital camera. 

The team tested this platform on COVID-19 samples and detected SARS-CoV-2 RNA within 30 minutes. Next, Merkx wants to apply this tool to rapidly detect sexually transmitted diseases to facilitate immediate treatment decisions in the clinic. 

References

1. van der Veer HJ, et al. ACS Cent Sci. 2023;9:657-667.
2. Kaminski MM, et al. Nat Biomed Eng. 2021;5:643-656.
3. Cherkaoui D, et al. Biosens Bioelectron. 2021;189:113328.