Development of loop-mediated isothermal amplification assay for rapid detection of cucurbit Leaf crumple virus

Authors: WALIULLAH, SUMYYA - University Of Georgia; Ling, Kai-Shu; CIENIEWICZ, ELIZABETH - Clemson University; OLIVER, JONATHAN - University Of Georgia; JI, PINGSHENG - University Of Georgia; ALI, EMRAN - University Of Georgia

Submitted to: International Journal of Molecular Sciences
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 3/2/2020
Publication Date: 3/4/2020
Publication URL: https://handle.nal.usda.gov/10113/6937443
Citation: Waliullah, S., Ling, K., Cieniewicz, E.J., Oliver, J.E., Ji, P., Ali, E. 2020. Development of loop-mediated isothermal amplification assay for rapid detection of cucurbit Leaf crumple virus. International Journal of Molecular Sciences. 21(5):1756. https://doi.org/10.3390/ijms21051756.
DOI: https://doi.org/10.3390/ijms21051756

Interpretive Summary: Cucurbit crops (i.e., cantaloupe, cucumber, pumpkin, squash and watermelon) are economically important vegetables and fruits in the U.S. In recent years, whitefly population has exploded in southern States where much of fall cucurbit crops are growing. Cucurbit leaf crumple virus (CuLCrV), a whitefly - transmitted virus has emerged as a major threat to the cucurbit crop productions in Southeastern States. Although some molecular-based detection tools are available for CuLCrV, there is still no simple and effective field-based detection method available. In collaboration with scientists from the University of Georgia and Clemson University, we have developed a loop-mediated isothermal amplification (LAMP) to achieve a simple, efficient and sensitive detection of CuLCrV in symptomatic and asymptomatic samples, thus is useful for early disease diagnosis in field settings.

Technical Abstract: A loop-mediated isothermal amplification (LAMP) assay was developed for simple, rapid and efficient detection of Cucurbit leaf crumple virus (CuLCrV), one of the most important begomovirus that infects cucurbits worldwide. A set of six specific primers targeting a 240 nt sequence region in the DNA A of CuLCrV were designed and synthesized for detection of CuLCrV from infected leaf tissues by real-time LAMP amplification using Genie® III system, which was further confirmed by gel electrophoresis and SYBR Green I DNA staining for visual observation. The optimum reaction temperature and time were determined, and no cross-reactivity was seen with other begomoviruses. The sensitivity assay revealed that LAMP was more sensitive than conventional PCR but less sensitive than qPCR. However, it was simpler and faster than the other assays evaluated. LAMP assay was also more efficient and sensitive in detecting CuLCrV from symptomatic and asymptomatic infected field samples than PCR. This simple, rapid, and sensitive method has the capacity to detect CuLCrV in samples collected in the field and is therefore suitable for early detection of the disease to reduce the risk of epidemics.