Development and Validation of a Real-Time PCR Assay for Rapid Detection of Two-Spotted Spider Mite, Tetranychus urticae (Acari: Tetranychidae).

Spider mites of the genus Tetranychus are difficult to identify due to their limited diagnostic characters. Many of them are morphologically similar and males are needed for species-level identification. Tetranychus urticae is a common interception and non-regulated pest at New Zealand's borders, however, most of the intercepted specimens are females and the identification was left at Tetranychus sp. Consequently, the shipments need to be fumigated. DNA sequencing and PCR-restriction fragment length polymorphism (PCR-RFLP) protocols could be used to facilitate the accurate identification. However, in the context of border security practiced in New Zealand, insect identifications are required to be provided within four hours of receiving the samples; thus, those molecular methods are not sufficient to meet this requirement. Therefore, a real-time PCR TaqMan assay was developed for identification of T. urticae by amplification of a 142 bp Internal Transcribed Spacer (ITS) 1 sequence. The developed assay is rapid, detects all life stages of T. urticae within three hours, and does not react with closely related species. Plasmid DNA containing ITS1 sequence of T. uritcae was serially diluted and used as standards in the real-time PCR assay. The quantification cycle (Cq) value of the assay depicted a strong linear relationship with T. urticae DNA content, with a regression coefficient of 0.99 and efficiency of 98%. The detection limit was estimated to be ten copies of the T. urticae target region. The assay was validated against a range of T. urticae specimens from various countries and hosts in a blind panel test. Therefore the application of the assay at New Zealand will reduce the unnecessary fumigation and be beneficial to both the importers and exporters. It is expected that the implementation of this real-time PCR assay would have wide applications in diagnostic and research agencies worldwide.

A HRM real-time PCR assay for rapid and specific identification of the emerging pest spotted-wing drosophila (Drosophila suzukii).

Spotted wing drosophila (Drosophila suzukii) is an emerging pest that began spreading in 2008 and its distribution now includes 13 countries across two continents. Countries where it is established have reported significant economic losses of fresh produce, such as cherries due to this species of fly. At larval stages, it is impossible to identify due to its striking similarities with other cosmopolitan and harmless drosophilids. Molecular methods allow identification but the current technique of DNA barcoding is time consuming. We developed and validated a rapid, highly sensitive and specific assay based on real-time PCR and high resolution melt (HRM) analysis using EvaGreen DNA intercalating dye chemistry. Performance characteristics of this qualitative assay, validation and applicability in a New Zealand quarantine framework are discussed. Application of this robust and independently validated assay across the spectrum of key food production and border protection industries will allow us to reduce the further spread of this damaging species worldwide.