Effective plant virus enrichment using carbon nanotubes and microfluidics.

Plant virus detection and identification in crops is a pillar for disease management, import of crop material, production of clean stock plants and basic plant virology studies. In this report, we present a platform for the enrichment and isolation of known or unknown viruses. This platform is based on carbon nanotube arrays inside a microfluidic device that can be a solution for the identification of low titer viruses from plants. Using our microfluidic devices, we achieved enrichment of two economically important viruses, the orthotospovirus, tomato spotted wilt orthotospovirus (TSWV) and the potyvirus, zucchini yellow mosaic virus (ZYMV). The carbon nanotube arrays integrated in these microfluidic devices are capable of trapping viruses discriminated by their size; the virus rich arrays can be then analyzed by common downstream techniques including immunoassays, PCR, HTS and electron microscopy. This procedure offers a simple to operate and portable sample preparation device capable of trapping viruses from raw plant extracts while reducing the host contamination.

Reverse transcriptase recombinase polymerase amplification for detection of tomato spotted wilt orthotospovirus from crude plant extracts.

Virus detection in early stages of infection could prove useful for identification and isolation of foci of inoculum before its spread to the rest of susceptible individuals via vectoring insects. However, the low number of viruses present at the beginning of infection renders their detection and identification difficult and requires the use of highly sensitive laboratory techniques that are often incompatible with a field application. To obviate this challenge, utilized Recombinase Polymerase Amplification, an isothermal amplification technique that makes millions of copies of a predefined region in the genome, to detect tomato spotted wilt orthotospovirus in real time and at the end point. The reaction occurs isothermically and can be used directly from crude plant extracts without nucleic acid extraction. Notably, a positive result can be seen with the naked eye as a flocculus made of newly synthesized DNA and metallic beads. The objective of the 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. Results can be obtained in situ without the need of sending the samples to a specialized lab.