Development of PCR tests for the identification of Diaspidiotus perniciosus (Hemiptera: Diaspididae)

The San Jose scale, Diaspidiotus (Quadraspidiotus) perniciosus (Comstock, 1881), is a significant economic pest of fruit crops regulated under the Common List of Quarantine Pests of the Eurasian Economic Union. In quarantine phytosanitary laboratories, diagnosis of this species is typically performed using morphological methods; PCR teats for D. perniciosus are not routinely employed. This article describes the development of PCR tests for the identification of D. perniciosus based on speciesspecific primers Dp1-F/Dp1-R, which amplify a 216 bp amplicon, and a Dp1-probe targeting a region of the mitochondrial COI gene. In silico testing of the primer pair and probe demonstrated their species specificity for D. perniciosus. Three PCR tests were developed: 1) with electrophoretic detection of PCR products; 2) a real-time PCR with the intercalating dye Sybr Green I; and 3) a real-time PCR using a TaqMan probe. They were tested on DNA samples of D. perniciosus and 15 other Diaspididae species, including eight species of the genus Diaspidiotus. In all three tests, a positive result was only obtained for the target species, demonstrating the specificity of the proposed identification methods. The analytical sensitivity of each of the three PCR tests, when tested on D. perniciosus DNA samples with concentrations ranging from 0.1 to 40.4 ng/μL, was 100%, indicating that the PCR tests can detect the target DNA at its minimum concentration. The developed PCR tests can be recommended for use in quarantine phytosanitary measures in laboratory practice for the identification of D. perniciosus, including at pre-imaginal stages; the choice of a test depends on the available laboratory equipment.

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