Development of a reverse transcription recombinase polymerase amplification combined with lateral flow assay for equipment-free on-site field detection of tomato chlorotic spot virus

Abstract

Abstract Background Tomato chlorotic spot virus (TCSV) is an economically important, thrips-transmitted, emerging member of the Orthotospovirus genus that causes significant yield loss mainly in tomatoes, but also in other vegetable and ornamental crops. Disease management of this pathogen is often challenging due to the limited availability of natural host resistance genes, the broad host range of TCSV, and the wide distribution of its thrips vector. Point-of-care detection of TCSV with a rapid, equipment-free, portable, sensitive, and species-specific diagnostic technique can provide prompt response outside the laboratory, which is critical for preventing disease progression and further spread of the pathogen. Current diagnostic techniques require either laboratory-dependent or portable electronic equipment and are relatively time-consuming and costly. Results In this study, we developed a novel technique for reverse-transcription recombinase polymerase amplification combined with lateral flow assay (RT-RPA-LFA) to achieve a faster and equipment-free point-of-care detection of TCSV. The RPA reaction tubes containing crude RNA are incubated in the hand palm to obtain sufficient heat (∼36 °C) for the amplification without the need for equipment. Body-heat mediated RT-RPA-LFA is highly TCSV-specific with a detection limit as low as ∼6 pg/μl of total RNA from TCSV-infected tomato plants. The assay can be performed in 15 min in the field. Conclusion To the best of our knowledge, this is the first equipment-free, body-heat-mediated RT-RPA-LFA technique developed to detect TCSV. Our new system offers a time-saving advantage for the sensitive and specific diagnostic of TCSV that local growers and small nurseries in low-resource settings can use without skilled personnel.