One Step, POC Sample to Answer Process for RNA Analysis Outside the Laboratory

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PROJECT SUMMARY 1 Lynntech has developed a process to readily identify specific genomic loci in RNA-based bacteriophage and 2 viruses. Although we developed a simple, automated sample preparation device (SPM) that will purify 3 genomic RNA for downstream processing, such as reverse transcription, we also demonstrated direct RT-PCR 4 amplification of a specific dengue locus using our convective units. This direct detection of dengue eliminates 5 the need for an independent sample preparation step in the diagnosis of dengue infection. In addition, we 6 have transitioned our convective amplification units to perform RNA reverse transcription (RT), as well as DNA 7 amplification (PCR). Lynntech's convective RT-PCR reaction has successfully identified genomic loci within the 8 MS2 bacteriophage, two strains of the Ebola virus, and the four serotypes of the dengue virus. The convective 9 assay is based on the principle that two different temperatures at the ends of a cylinder will result in a 10 buoyancy-driven steady circulatory flow between those ends. Thus, PCR reagents in the cylinder will flow 11 through a temperature gradient allowing the necessary steps for amplification: denaturation, annealing, and 12 elongation. Convective amplification is rather appealing in that it requires very little power. Because reagents 13 circulate within a temperature gradient, there is no need for temperature ramping and power is not needed to 14 cool, and then heat, the reaction. So the convective system can be powered by batteries and can provide a 15 portable means to perform RT-PCR at the point-of-care. In addition, this portable RT-PCR device can be quite 16 inexpensive. 17 Lynntech has demonstrated the reverse transcription and subsequent cDNA amplification of MS2, Ebola, and 18 dengue RNA, using convective RT-PCR. Our data indicated excellent specificity for both the Ebola virus and 19 the dengue virus. Notably, for the dengue virus, we were able readily distinguish the four serotypes in our 20 convective RT-PCR assay, despite the fact that the genomes of these four serotypes share 60-70% homology. 21 In addition, our convective RT-PCR assay was quite sensitive. We were able to easily detect less than 100 22 genomic copies of the dengue 3 virus in our assay. This would equate to less than 1 µL of plasma from an 23 infected patient. Indeed these data underscore the applicability of our convective assay to the diagnosis of 24 dengue infection at the point-of-care in resource-limited regions of the world. 25 During Phase II of this program, Lynntech will further develop our convective amplification system into a 26 single, portable, one-step unit that can be used beyond the confines of the traditional laboratory to diagnose 27 dengue infection, as well as to study RNA processes. We will transition this system to a multiplex assay that 28 will identify the four dengue serotypes in a single reaction. This assay will combine our portable genome 29 amplification module with a sophisticated gold-nanoparticle-based lateral flow system to provide a point-of-care 30 detection and identification assay for the dengue virus.