Qualitative real-time PCR identification of the khapra beetle, Trogoderma granarium (Coleoptera: Dermestidae)

Abstract

The identification of insect species is generally achieved by analyzing the specific morphological characteristics or, in more recent years, by polymerase chain reaction (PCR) assays of DNA sequences. PCR assays, which detect specific segments of DNA, have been established as a highly sensitive method for a wide range of applications, including the verification of hereditary diseases and for criminal investigations. For the identification of insects, the CO1 (cytochrome c oxidase subunit I) gene of mitochondrial (mt) DNA is often used. However, analyses of the entire mtDNA sequence might yield more reliable regions for use in the identification of insect species. Here we developed a method for real-time PCR identification of the khapra beetle, Trogoderma granarium Everts, by comparing the whole mtDNA sequences of three species of dermestid beetle: the khapra beetle, the varied carpet beetle, Anthrenus verbasci (L.), and the black carpet beetle, Attagenus unicolor japonicus Reitter. Several khapra beetle-specific mtDNA regions were identified and selected for use in the real-time PCR identification of these beetles based on Taq-Man® chemistry. Specificity tests conducted with the DNAs of several insects, including those of the three beetles described above, revealed that the primer/probe sets designed for the real-time PCR were highly specific to their targets.