A Real-time PCR assay for rapid identification of Helicoverpa armigera (Lepidoptera: Noctuidae) from closely related species

The bollworm, <em>Helicoverpa armigera</em> is one of the most important agricultural pests in the world. Diagnosis of this species morphologically is difficult from the similar species <em>H. assulta</em>, <em>H. punctigera and H. zea </em>as the adults are identified based on genitalia morphology and larval identification is limited due to shared morphological characters. To provide an accurate diagnosis of New Zealand border interceptions we have developed and validated a real-time PCR assay to identify <em>H. armigera </em>from commonly occurring exotic <em>Helicoverpa</em> species, <em>H. assulta</em> and <em>H. zea</em>. The newly designed primers and probe were optimised, and the assay demonstrated its specificity to <em>H. armigera</em> with no false positives observed in the non-target species tested in this study. The specificity of the assay was tested against 64 samples of Lepidoptera from 14 countries including 48 <em>Helicoverpa </em>samples. The sensitivity of the assay showed it can detect up to 1 copy /µL of the DNA fragment. Therefore, this assay can be used for rapid detection of <em>H. armigera</em>, which is imperative in quarantine application to determine the presence of exotic species regardless of its life stage.