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

Abstract

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.

Figure 1. Real-time PCR assay for the identification of Drosophila suzukii primer pair alignment with closely related species.

There is a mismatch in the reverse primer target sequence match at the 3′ end and non-target sequence D. subpulchrella (G → A), due to SNP-1. An additional mismatch was introduced in the reverse primer at third base from the 3′ end (A → C) to further suppress the binding of D. subpulchrella.

Figure 2. Species differentiation using the melting profile and high resolution melt analysis.

A, Melting profiles of the species amplified by the D. suzukii assay primer pair Dsuz1F – Dsuz6R. Green  =  D. suzukii, Red  =  D. subpulchrella and Blue  =  D. biarmipes, other species did not amplify and had flat melt curves. B, Melt curve difference plots comparing Green  =  D. suzukii and Red  =  D. subpulchrella melt curves.

Figure 3. Efficiency of the real-time PCR assay for the identification of D. suzukii.

Plasmid dilution series were used to create calibration curves for efficiency calculations. Top panel shows the amplification curves, threshold = 100 raw fluorescence units (rfu) and coloured lines indicate the Cq (rfu threshold cycle) values for each of the dilution series replicates. Bottom panel shows the standard curve built from Cq (threshold cycle) values against the log copy number (range = 10⁷−10² copies). The 95% confidence interval of the slope is plotted in red lines and the r² = 0.998. The fit of the slope was also optimised using the Akaike Information Criteria (AIC) and the AIC statistic = 9.52.