Closed-Tube pH-Sensitive Loop-Mediated Isothermal Amplification for Rapid Detection of Tomato Leaf Curl New Delhi Virus in Vegetable Crops

Tomato leaf curl New Delhi virus (ToLCNDV) threatens cucurbit and solanaceous vegetable production, highlighting the need for rapid screening outside centralized laboratories. Here, we developed a closed-tube, pH-sensitive loop-mediated isothermal amplification (pH-LAMP) assay targeting a conserved region of ToLCNDV DNA-A. The assay incorporated six primers into a 25-µL reaction containing a weakly buffered master mix and a pH indicator. Incubation at 65 °C for 40 min produced a red-to-yellow color change that could be interpreted visually without opening the reaction tube. In a ten-fold dilution series of a ToLCNDV DNA-A plasmid, positive reactions were observed at concentrations ranging from 1000 ng µL−1 to 1 fg µL−1, with 1 fg µL−1 being the lowest tested concentration yielding a positive result. No cross-reactivity was observed with the five non-target vegetable viruses tested, and the healthy wax gourd control remained negative. In field screening, all 17 samples selected on the basis of symptoms from seven crop species tested positive by both pH-LAMP and conventional PCR, yielding 100% positive agreement in this selected symptomatic sample set. An additional 16 asymptomatic samples collected from plants of four crop species growing near symptomatic plants were screened, of which 11 yielded positive pH-LAMP results. These proof-of-concept results support the potential of closed-tube pH-LAMP for rapid, preliminary ToLCNDV screening using portable heating equipment and visual readout without post-amplification tube opening.