Detection of Tomato spotted wilt orthotospovirus from crude plant extracts using Reverse Transcriptase- Recombinase Polymerase Amplification in endpoint and real-time

Abstract

AbstractVirus 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, 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 Tomato spotted wilt orthotospovirus. The reaction occurs at 39 ℃ and can be used directly from crude plant extracts without nucleic acid extraction. Notably, positive results 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.