Multiplex polymerase chain reaction assay for pest identification of rice green leafhoppers ( Nephotettix , Hemiptera: Cicadellidae)

Abstract Species-specific primers were designed from reference sequences of Nephotettix cincticeps (Uhler), Nephotettix malayanus Ishihara et Kawase, Nephotettix nigropictus (Stål), and Nephotettix virescens (Distant) (Hemiptera: Cicadellidae) and applied in multiplex polymerase chain reaction (PCR) for the molecular identification. The assay yielded distinct amplicons of 263 bp for N. malayanus, 320 bp for N. virescens, 401 bp for N. cincticeps, and 502 bp for N. nigropictus, enabling clear resolution by agarose gel electrophoresis. This method provides a reliable and efficient approach for field surveys and population monitoring of rice green leafhoppers, and is expected to greatly reduce misidentification while providing robust support for pest management decision-making.