A Real-Time PCR Assay for the Separation of Autographa gamma (Noctuidae: Plusiinae) From Morphologically Similar Species in North America

Abstract

The silver Y moth, Autographa gamma L. (Noctuidae: Plusiinae), is a pest of major economic importance in its native range of Europe, Asia, and North Africa. Although not present in North America, larvae of A. gamma are commonly intercepted in commodity shipments at U.S. ports, and adult surveys are conducted each year in more than 20 states. Because of the similarity of A. gamma to several native North American species that are attracted to the same pheromone lure, morphological identification of adults is difficult and requires dissection. In 2010, a specimen of Autographa californica (Speyer, 1875) (Lepidoptera: Noctuidae) from Pennsylvania was incorrectly identified as A. gamma, signaling the need for an alternative method of rapid identification. Here we detail a real-time PCR assay capable of identifying A. gamma specimens in approximately 45 min using extracted DNA. The assay uses a hydrolysis probe that targets a species-specific segment of the CO1 DNA barcode region, while a control probe targets a conserved region of 18S rDNA. The assay was tested with two independent runs of 452 specimens of Plusiinae representing 23 different species. The assay provided unambiguous data 99.7% of the time and did not result in any false positives; these data were used to develop a rule set for interpreting the real-time PCR results. In addition, the same diagnostic probe was tested in bulk sample simulations using real-time PCR and droplet digital PCR where A. gamma could be detected in concentrations as low as 1:1,000,000 (gamma:californica). These experiments provide baseline data for developing a bulk sample assay.

Assay Optimization Can Equalize the Sensitivity of Real-Time PCR with ddPCR for Detection of Helicoverpa armigera (Lepidoptera: Noctuidae) in Bulk Samples

“…While the use of ddPCR is an attractive method for pest detection in bulk samples due to its accuracy, tolerance to PCR inhibitors, and method of direct standard-free quantification [ 25 ], it is costly, and the systems are not widely available. Conversely, real-time PCR, because of the relatively low cost of machine ownership and operation, is frequently utilized for detection of pest species of many types (e.g., [ 26 , 27 , 28 ]). Unfortunately, the real-time PCR-based methods for detecting H. armigera in bulk samples do not perform well in real-world sized samples which routinely include hundreds of specimens [ 24 , 29 ].…”

Viral Prevalence and Genomic Xenology in the Coevolution of HzNV-2 (Nudiviridae) with Host Helicoverpa zea (Lepidoptera: Noctuidae)

“…Positively identified specimens of H. armigera, H. zea, and lab-reared hybrids between these two species were used as controls. Genomic DNA was extracted from the terminal segment of adult abdomens using a Qiagen DNeasy Blood and Tissue Kit (Qiagen, Valencia, CA, USA) following the protocol described in Tembrock et al [50]. DNA concentration and purity were measured on a NanoDrop 2000 Ver.…”