Integrated Diagnostic Approach Using qPCR, ddPCR and LAMP-Based Molecular Tools for Two Leafhopper Species Identification (Nephotettix cincticeps and Nephotettix virescens)

Abstract

Accurate identification of the leafhopper species Nephotettix cincticeps and N. virescens is necessary because they feed on phloem sap and transmit plant pathogens including viruses that can cause significant damage to rice plants. Initially, we examined their physical traits using a microscope and a specific camera and observed differences between the two species. To further validate these distinctions on a molecular level, we designed primers based on mitochondrial genome sequences obtained from the NCBI database. The primers were effectively used in general PCR and loop-mediated isothermal amplification (LAMP) tests. General PCR was successfully performed at 61°C using NC_mt5117F33 and NC_mt5438B33 primers for N. cincticeps and NV_mt3089F31, NV_mt3360B31 primers for N. virescens. The effectiveness of the selected primers was validated using quantitative PCR (qPCR) on the pooled gDNA samples and droplet digital PCR (ddPCR). The best conditions for LAMP were 61°C and time 45 min, resulting in the most favourable amplification outcomes. Additionally, LAMP enables diagnosis at the individual level in the field, and qPCR and ddPCR can be used for relative or absolute quantification, even from pooled gDNA from multiple species. This study emphasises the importance of combining traditional and molecular techniques in primary pest species diagnostics to ensure precise and timely pest control, which can contribute to improved pest management strategies.