RNA Detection Assay (Exemplified with an Assay for SARS-CoV-2)

University of St. Andrews Background
A new assay for detection of any desired RNA molecule has been developed that utilizes the type III CRIPSR defence system. The assay system comprises Cas10, a molecule capable of binding a target nucleic acid, a nuclease, a reporter system and optionally ATP.
A novel assay to detect Covid 19 with a low limit of detection has been developed and the assay can be adapted to detect other pathogens of interest. With modifications and suitable design it is also possible to detect single nucleotide polymorphisms (SNPs).
Technology Overview
The assay described provides a step change improvement to existing assays. (See advantages below).
Current detection methods include:
Reverse Transcriptase Polymerase Chain reaction (RT-PCR)
The most widely used method utilizes the reverse transcription polymerase chain reaction (RT-PCR).
RNA is extracted from the sample and converted to cDNA using reverse transcriptase and then amplified using a specific primer.
Amplification is measured in real time using a fluorescent dye or a combination of a quencher and a sequence specific DNA probe labelled with a fluorescent molecule.
Potential disadvantages – may not distinguish between intact RNA whole virus particles from viral RNA which may result in false positives. The method also requires access to expensive laboratory instruments and skilled laboratory. The approximate assay time is 1-2h after viral RNA extraction.
Isothermal nucleic acid amplification methods
Allows amplification of the target gene sequence at a constant temperature without the requirement for thermal cycling equipment (£2000-3000). Still requires amplification step. Faster than RT-PCR with approx. assay time of 30min
Digital PCR
Potential advantages over quantitative PCR in that calibration curves are not required, higher precision, less susceptible to artefacts that may arise from sub optimal amplification efficacy because of PCR inhibitors or primer/template mismatch. Has higher analytical sensitivity but Digital PCR requires longer hands-on time leading to higher staff costs and still requires high equipment and consumable costs
CRISPR based assays
Recognises foreign RNA or DNA material in a sequence specific way and comprises one or more CRISPR-associated endonuclease (Cas) systems. Requires base paring between the guide RNA and the target sequence. Paves the way for CRISPR based diagnostic assays
The assay requires two steps. SHERLOCK is based on the amplification of the target DNA or RNA (RNA detection requires RT-PCR followed by Cas 13 mediated detection using colorimetric or fluorescent readouts). Limit of detection 42 copies /reaction.
DETECTR utilizes Cas12
SHERLOCK has a reaction time of approximately 1 hour, 100% specificity and sensitivity in EUA clinical evaluation, utilises standard laboratory equipment and a microplate reader and stated limit of detection of 6.75cp/ul
A recent review (JClin Microbiol59, e00745-20) highlighted advantages and potential limitations of these assays. See www.ncbi.nlm.nih.gov/pmc/articles/PMC8106734/pdf/JCM.00745-20.pdf
Other methods used include microarray and next generation sequencing. However, the researchers do not consider that these methods are suitable for routine diagnostic testing.
A full review of molecular diagnostics assays for Covid 19 was published in February 2021 (https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7898297/)
Stage of Development
TRL3 – proof of concpet has been established. A pre-print describing the work is available at: https://doi.org/10.1101/2021.09.13.460032

No requirement for an amplification step using Reverse Transcriptase Polymerase Chain Reaction (RT-PCR)
The assay comprises two intrinsic signal amplification steps
Does not require RNA extraction from samples (although could be undertaken to further improve the limit of detection if required)
Limit of detection approx. 1 fM
Use of NucC nuclease (rather than alternative nucleases) as NucC is activated by lower concentrations of cyclic nucleotides, enhancing assay sensitivity
Nuclease employed has negligible background activity in the absence of cyclic nucleotide and unlike alternative nucleases does not degrade its cyclic nucleotide activator
The NucC nuclease cleaves DNA to generate the signal output – avoiding problems with instability of RNA seen with competing approaches
Potential advantages over other assay technologies in relation to overall assay time

This new method approach to RNA detection is not currently comercially available. Primary application would be human clinical diagnostics. Other potential areas would include veterinary applications and identification of crop pathogens.
Potential applications – RNA detection methods, clinical diagnostics, Covid 19, SARS-CoV-2, CRISPR Cas 10, pathogen detection.
Translational/development collaboration and/or licence. The University of St Andrews envisage that the collaboration would involve further laboratory validation followed by commercial development of a diagnostic assay for SARS-CoV-2 and other organisms of interest.

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