Development and Application of a Multiplex PCR Assay for Simultaneous Detection of Tomato Yellow Leaf Curl Virus and Tomato Leaf Curl New Delhi Virus

Abstract

Tomato leaf curl New Delhi virus (ToLCNDV) and tomato yellow leaf curl virus (TYLCV) are two important viral pathogens that severely affect Solanaceae and Cucurbitaceae plants. In order to reduce the further spread of these viruses, it is crucial to establish an efficient and reliable method to accurately detect the viruses. In this study, a multiplex PCR assay for the simultaneous detection of TYLCV and ToLCNDV was established. Three primer pairs designed from conserved regions within the coat protein or movement protein-encoding regions of the respective viruses were employed in the assay. The optimization of parameters such as primer concentration was set at 0.15 μM/0.15 μM, 0.25 μM/0.25 μM, and 0.50 μM/0.50 μM for ToLCNDV-DNA-A-F/R, TYLCV-F/R, and ToLCNDV-DNA-B-F/R primer pairs. At optimal primer concentrations, the multiplex PCR method demonstrates effective performance with an annealing temperature ranging from 51 °C to 66 °C. The specificity of the assay evaluated by testing against other begomoviruses showed no evidence of cross-amplification. Further sensitivity analysis performed using a serially diluted plasmid containing viral targets as templates demonstrated high sensitivity with a detection limit of 103 copies/μL. Field surveys utilizing the multiplex PCR assay successfully identified the infection of TYLCV and ToLCNDV in field-collected samples.