This project covers a number of technical areas; design and development of the novel instrument and reaction vessel, development of the reagent including the enzyme, the buffer system and it’s lyophilization, assay development and validation, finally a performance evaluation for the complete platform. The commercial partner has developed the instrument and reaction vessel from concept to Alpha prototype entirely within the duration of the KRONO project. A new injection mould has been developed making the reaction vessels for the assays, including development of sealing means to ensure that the lyophilized reagents can be stored correctly and a barcoding system, such that the instrument can be programmed by scanning the vessel. In terms of the instrument this has involved the complete design of the electronics, mechanics and hardware of the system from the ground up. BG research have taken on additional staff to complete and have developed new software, firmware, electronics and the prototype instrument within the duration of the programme of work. The current status is that a set of Alpha prototype instruments have been built and are currently undergoing final PCR testing prior to shipment to partner labs in early February. User manuals and training manuals for these have been developed. Using the prototype BioGene demonstrated direct detection of a SARS CoV-2 at below 5,000 virions/ml direct from a contrived nasal swab sample using a lyophilized version of the reagent, passing the originally stated aim of detection at below 10,000 virions/ml of nasal swab eluate. In terms of the reagent, a method of producing the enzyme at scale has been developed and validated. A final version of the buffer was developed and validated using live SARS CoV-2 virus and archived patient samples. The reagent was lyophilized and demonstrated identical sensitivity to the frozen reagent, indicating that the project goal of portable diagnostics could be supported. The novel reagent was tested with saliva and it was possible to detect infection from unprocessed saliva. With regard to the assay itself, initial studies were performed with frozen reagents and a singleplex N gene assay. As new variants emerged the team moved to an N gene and RdRp duoplex assay, demonstrating a lower limit of detection below 5 virions per reaction using a surrogate virus. An internal positive control was developed and shown to not negatively impact the target lower limit of detection for the assay. To support future use in emerging outbreaks an assay was developed to detect “disease X”, in this case the direct from blood detection of yellow fever. An assay was developed and the migration to direct detection from blood is underway and this assay will be validated shortly. In terms of SARS CoV-2 INMI have performed live viral culture testing and showed that the virus is lysed and rendered amplifiable by the reagent and that the assay detects the pathogen to below 40 actual virions per reaction, the assays have been validated at the addition of 10µl of crude sample per reaction and meet the originally outlined performance criteria. The WHO have developed a target product profile for COVID stating that simple, low-cost tests must be able to detect to below 10,000 virions/ml and as such this technology can easily meet this requirement.
