A Loop-mediated Isothermal Amplification Method for White-backed Planthopper-specific Detection

摘要

A loop-mediated isothermal amplification (LAMP) primer set (WBPH-65) was designed for the species-specific detection of white-backed planthopper (WBPH) Sogatella furcifera based on the full-length sequence of the internal transcribed spacer 2 (ITS2) (KC417469.1). The WBPH-65 primer set consists of six primers (total 165 bp), F3 (18 bp), B3 (18 bp), FIP (43 bp), BIP (40 bp), LF (21 bp), and LB (25 bp). After the LAMP reaction of three rice planthoppers, S. furcifera, Nilaparvata lugens, and Laodelphaxstriatellus, with the WBPH-65 primer set for 60 min at 65 °C, the LAMP products were observed in the genomic DNA of S. furcifera only. According to the DNA amount of S. furcifera and incubation duration at 65 °C, the difference of fluorescence relative to the negative control (0 ng) was clearly observed in a 40-rnin incubation with 10 and 100 ng or in case of 60-min incubation with 0.01, 0.1,1,10, and 100 ng. There was little difference in fluorescence between the negative control and all the other DNAs tested in 20- and 30-min incubations. On the other hand, the WBPH-65 primer set without LF and LB primers showed little amplification in the genomic DNAs of the three rice planthoppers, S. furcifera, N. lugens, and L. striatellus in a 60-min incubation. These results suggest that all six primers (F3, B3, FIP, BIP, LF, and BF) are necessary for the WBPH-65 primer set to detect S. furcifera within a 60-min incubation, and is able to discriminate S. furcifera from at least N. lugens and L. striatellus.