Abstract:
Molecular species identification is becoming more wide-spread in diagnostics and ecological studies, particularly with regard to insects for which morphological identification is difficult or time-consuming. In this study, we describe the
development and application of a single-step multiplex PCR for the identification
of three mealybug species (Hemiptera: Pseudococcidae) associated with grapevine
in South Africa: Planococcus ficus (vine mealybug), Planococcus citri (citrus
mealybug) and Pseudococcus longispinus (longtailed mealybug). Mealybugs are
pests on many commercial crops, including grapevine, in which they transmit viral
diseases. Morphological identification of mealybug species is usually timeconsuming,
requires a high level of taxonomic expertise and usually only adult females can be identified. The single-step multiplex PCR developed here, based on the mitochondrial cytochrome c oxidase subunit 1 (CO I) gene, is rapid, reliable, sensitive, accurate and simple. The entire identification protocol (including DNA extraction, PCR and electrophoresis) can be completed in approximately four hours. Successful DNA extraction from laboratory and unparasitized fieldcollected individuals stored in absolute ethanol was 97%. Specimens from which DNA could be extracted were always correctly identified (100% accuracy). The
technique developed is simple enough to be implemented in any molecular laboratory. The principles described here can be extended to any organism for which rapid, reliable identification is needed.
