SYBR green-based real-time PCR test for the identification of adults and larvae of the Japanese beetle Popillia japonica newman (Coleoptera Scarabaeidae).
The Japanese beetle (Popillia japonica Newman) (Coleoptera Scarabaeidae) is a polyphagous invasive pest included in quarantine lists in Europe and in many countries of Africa, Asia and America. No specific molecular methods were available for the identification of adults and preimaginal stages. To bridge this gap, a rapid method (<2 h) based on SYBR Green real-time PCR for identifying larvae and adults of P. japonica was developed in this study. SYBR Green chemistry was chosen for its cost-effectiveness and adaptability although it has a lower specificity than other real-time PCR tests such as the as TaqMan test which requires a probe design. The in-silico primer specificity was tested, and the theoretical amplicon did not match any of the sequences deposited in databanks. The diagnostic specificity (exclusivity) of the test was assessed by testing the method on taxonomically related species (Popillia anomaloides Kraatz, P. hilaris Kraatz and P. fallaciosa Fairmaire), other Coleoptera and unrelated insect species, with no cross-reactions. Performance characteristics of the test, in terms of diagnostic specificity (inclusivity), analytical sensitivity, and accuracy, were evaluated. The analytical sensitivity determined on a single larva showed a LoD value of 3.20 pg/µL. The molecular test developed could be highly beneficial for the phytosanitary controls at entry points for the regulatory confirmation of specimen identity and/or for monitoring the presence and spread of P. japonica in the field.