Construction of an infectious clone of tomato leaf curl New Delhi virus and development of a duplex assay through recombinase polymerase amplification-lateral flow dipstick to detect the virus and tomato yellow leaf curl virus simultaneously

The co-infection of tomato yellow leaf curl virus (TYLCV) and tomato leaf curl New Delhi virus (ToLCNDV) in the same host complicates disease diagnosis and management, not to mention that each virus alone can cause devastating losses to its respective host crops across many tomato- and cucurbit-growing areas. However, diagnosis of mixed viral infections in the same host plants can be tedious, costly and challenging. This study aims to establish a rapid, sensitive, and specific duplex recombinase polymerase amplification-lateral flow dipstick (RPA-LFD) assay to simultaneously detect TYLCV and/or ToLCNDV in co-infected samples. We constructed an infectious clone from a ToLCNDV strain of the Asian clade isolated from a diseased cucumber plant in China, and confirmed that it could co-infect Nicotiana benthamiana together with an infectious clone of the TYLCV-IL strain, providing the defined viral inoculum for method development and validation. An optimal duplex RPA-LFD assay was developed by screening specific RPA primers and probes targeting conserved sequences of both viruses and optimizing reaction parameters, which produced interpretable results directly on a test strip after 20 min at 39 °C. With the purified plasmid DNA, the analytical limits of detection of the duplex RPA-LFD for TYLCV and ToLCNDV were 102 and 101 copies/μL, respectively. No cross-reactions were detected against three other begomoviruses. Further validation using total DNA from co-inoculated N. benthamiana and field-collected samples showed high consistency with conventional PCR, and the assay effectively distinguished single-virus from mixed infections. This duplex RPA-LFD assay provides a reliable diagnostic for detecting and surveying co-infections caused by TYLCV and ToLCNDV.