POSTERS: Pathogen detection, quantification and diagnosis
Ultrasensitive and in-situ detection of a plant virus by a nanotube-filtering device and isothermal amplification
Juan Francisco Iturralde Martinez - The Pennsylvania State University. Nestor Perea-Lopez- The Pennsylvania State University, Cristina Rosa- The Pennsylvania State University, Mauricio Terrones- The Pennsylvania State University, Edwin Rajotte- The Pennsylvania State University
Plant virus detection at the site of initial infection could prove useful for identification prior to transmission by vectoring insects. This would allow for earlier implementation of a management system and could prevent epidemics. However, the low viral titer during early infection renders detection and identification difficult and requires the use of highly sensitive laboratory techniques that are often incompatible with field application. To obviate this challenge, we designed a Recombinase Polymerase Amplification, a molecular technique that makes millions of copies of a predefined region in the genome, in this case of the tomato spotted wilt orthotospovirus (TSWV) genomic segment M. The reaction occurs at 39 ? and can be used directly from crude plant extracts without nucleic acid extraction. A positive result can be seen with the naked eye as a flocculus made of newly-synthesized DNA and metallic beads. This technique can be coupled with the use of our nanotube-based microfluidics devices to trap viruses by size and to concentrate them at detectable levels for any downstream technique. The objective of the combined procedure is to create a portable and affordable system that can isolate and identify viruses in the field, from infected plants and suspected insect vectors, and can be used by scientists and extension managers for making informed decisions for viral management.