Development of PCR, LAMP and HRM-Based Molecular Species Diagnostic Tools for Identifying Four Agricultural Pests in the Genus Helicoverpa (Lepidoptera: Noctuidae)

Abstract

Within the subfamily Heliothinae, the genus Helicoverpa comprises several highly destructive agricultural pests, including H. armigera, H. assulta, H. punctigera and H. zea. These species cause significant global crop losses due to their wide host range, long-distance migratory behaviour, and potential for interspecific hybridization. Their close morphological resemblance poses major challenges for accurate identification. Therefore, the development of rapid and reliable molecular diagnostic tools is crucial for effective monitoring and management. For primer design, complete mitochondrial genome sequences of the four Helicoverpa species were aligned to identify species-specific regions, primarily within the COX1–COX2 fragment. Species-specific PCR assays were optimised and successfully distinguished all four species. The same primer regions were adapted for LAMP by designing the outer primers (F3 and B3) and adding inner (FIP, BIP) and loop (LF, LB) primers. LAMP assays were highly effective for H. armigera and H. assulta, allowing field-ready detection within 20 and 25 min, respectively, at 62°C. While PCR and HRM distinguished all four species, LAMP assays were successfully optimised only for H. armigera and H. assulta. Additionally, an HRM assay was developed and validated, enabling efficient large-scale discrimination of Helicoverpa species based on distinct melting profiles. This study presents robust PCR, LAMP and HRM-based molecular diagnostic tools for the rapid and accurate identification of four major Helicoverpa pest species: H. armigera, H. assulta, H. punctigera and H. zea. These assays provide reliable, cost-effective and scalable solutions for species-specific detection and can be readily integrated into regional and international Helicoverpa pest monitoring and management programmes.