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A multiplex PCR assay for the simultaneous identification of three mealybug species (Hemiptera: Pseudococcidae)

Published online by Cambridge University Press:  13 December 2007

D.L. Saccaggi ,
K. Krüger  and
G. Pietersen
D.L. Saccaggi
Affiliation:
Department of Zoology and Entomology, University of Pretoria, Pretoria 0002, South Africa
K. Krüger*
Affiliation:
Department of Zoology and Entomology, University of Pretoria, Pretoria 0002, South Africa
G. Pietersen
Affiliation:
Citrus Research International, c/o Department of Microbiology and Plant Pathology, University of Pretoria, Pretoria 0002, South Africa
*
*Author for correspondence Fax: +27 12 362 5242 E-mail: [email protected]
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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 time-consuming, 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 field-collected 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.

Keywords

insectmealybugmultiplex PCRdiagnostic molecular identification
Type
Research Paper
Information
Bulletin of Entomological Research , Volume 98 , Issue 1 , February 2008 , pp. 27 - 33
Copyright
Copyright © Cambridge University Press 2008

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