Poster Y13 - A Simplified Purpose-Built Platform and Improved Diagnostic Tool for Testing of Endemic and Foreign Animal Vesicular Diseases

Introduction: The rapid and real-time sequencing brought about by nanopore technology can serve as an excellent molecular diagnostic tool to evaluate and combat threats to US agriculture and livestock. However, the portability and ease of use afforded by these small sequencing devices is often hampered by sample preparation requirements such as equipment that typically isn’t found outside of a laboratory setting. Additionally, sample preparation in the field adds significant risk of contamination during user operation, limiting diagnostic quality.

This study evaluated a semi-automated, field deployable system for the rapid handling of viral samples including RNA extraction, library preparation, and sequencing using the Oxford Nanopore MinION device. At the heart of the platform is a pump operated microfluidic device which facilitates sample amplification and library preparation in a closed system, significantly minimizing risk of contamination. This device incorporates multiple reaction and mixing chambers and has been expanded and optimized to simultaneously process up to four samples with minimal crossover, reducing the reagent cost and library production time. The device utilizes specially designed built-in structures for passive mixing as well as paramagnetic beads controlled by a magnet for active mixing. Along with adding increased mixing capabilities to the device, the paramagnetic beads also allow for isolation and precise movement of nucleic acids on-chip for sample control beyond what the automated syringe pumps offers. All the device inlets have been designed with integrated bubble traps to limit undesirable diffusion into the device and minimize sample loss from gaseous expansion during heating.

Materials and

Methods: The endemic Senecavalley Virus A (SVA) was used to model pathogenic FMDV. Loop mediated isothermal amplification (LAMP) was utilized to increase starting quantity of RNA recovered from nucleic acid extraction protocols in order to meet minimum requirements of tagmentation based library preparation. A multiplex LAMP primer set (6 total primer sets) was designed to evenly amplify 3 distinct regions covering 16% of the 7300bp genome without necessitating a thermocycler. All heating on the device is facilitated by a simple hot plate. SVA viral RNA was extracted following culture in LC cells. Vesicular fluid samples were swabbed from oral lesions found in SVA positive pigs.

Results, Conclusions, and Discussions: Using SVA isolates, our device successfully amplified all three target regions simultaneously and produced libraries with an alignment rate of 51% with a barcoding success rate of 93%. These generated libraries produced enough aligned reads to provide positive diagnosis within the first 27 minutes of sequencing across all three targeted regions. The generated libraries were able to identify single nucleotide variations within samples with 100% accuracy on highly enriched mutations (>20% presence). We then applied our system to vesicular lesion swabbed samples from SVA positive pigs. A total of 7 positive samples were tested along with three healthy controls, and device generated libraries were able to generate positive identifications in all three distinct regions based on both total distinct target reads (>45 per region) and alignment rate (>20%) within an hour of sequencing. The device has been expanded to process 4 samples simultaneously on a single microfluidic chip, with pump operation fully automated.

Acknowledgements (Optional):