آزمون TaqMan Real-Time PCR برای شناسایی سریع Agrotis segetum (Lepidoptera: Noctuidae)

The turnip moth, Agrotis segetum Denis and Schiffermaller (Lepidoptera: Noctuidae), is one of the most serious agricultural pests occurring throughout Iran. Pheromone/light traps are tremendously straightforward and well-liked methods for collecting and monitoring adult male noctuid moths particularly in regions where it is prudent to monitor for destructive insect pests. As these traps may attract more than one species and samples often degrade as adult moths typically remove a large portion of their scales while fluttering around inside the traps, morphological identification of collected noctuid species might be extremely strenuous. Furthermore, the early detection of A. segetum in different agro-ecosystems is of crucial for implementing any control strategies. Moreover, for efficient diagnosis of adults captured during domestic surveys and other immature stages intercepted at ports of entry, means of identification other than morphology are required. In such situations, using a molecular diagnostic protocol might be very useful especially when dealing with specimens that are in poor condition. Although most of the currently used molecular methods such as DNA barcoding allow identification, they are time consuming. We have developed a real-time PCR assay based on TaqMan for distinguishing the turnip moth from other noctuids might be found in vegetable fields. A A. segetum-specific set of primers and probe were designed within the mitochondrial cytochrome oxidase I (COI) gene using AlleleID 7 software. Total genomic DNA was extracted from the legs of adult specimens using DNeasy Blood and Tissue Kit (Qiagen) according to manufacturer's instructions. All reactions were performed on an ABI7300 real-time PCR instrument. The specificity of the assay was assessed using 5 species of other Agrotis species (A. ipsilon (Hufnagel, 1766), A. exclamationis Campion, 1915, A. obesa Razowski, 2013, A. sardzeana Brandt, 1941, A. trux (Hübner, 1824)) commonly occur in Iran. Moreover, Insilco testing was also used to further test the specificity. The samples were also tested using a TaqMan 18S internal control real time PCR (Applied Biosystems, USA) in duplex (with both control and diagnostic primers and probes). No interactive or negative effects of multiplexing the two probe systems were observed. All A. segetum specimens were detected, and no cross reactions with other Agrotis species were detectd. The method was also tested on larvae and pupae and proved to be applicable for both those immature stages of A. segetum. In conclusion, as a quick, accurate and specific alternative to morphology or conventional PCR-based methods, the TaqMan qPCR assay developed here saves precious time and provides a useful tool to aid in diagnosis of A. segetum and is appropriate for identification of this pest especially when a large number of insect specimens are to be processed.