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Real‐time PCR assays for rapid detection of Zeugodacus cucumis and Bactrocera jarvisi (Diptera: Tephritidae) for quarantine application

Li, Dongmei, Nair, Sreelakshmi, Anderson, Diane, Doddala, Prasad, Gunawardana, Disna N., George, Sherly
Journal of applied entomology 2019 v.143 no.1-2 pp. 155-163
Bactrocera, Cucumis, DNA, Zeugodacus, binding sites, fruit crops, fruit flies, fruits, horticulture, immatures, industry, monitoring, pests, protocols, quantitative polymerase chain reaction, quarantine, rapid methods, vegetable crops, New Zealand
Zeugodacus cucumis and Bactrocera jarvisi are pests of fruit and vegetable crops and cause damage to horticulture industries. Immature stages of these two fruit fly species have been intercepted in New Zealand a number of times. Identification to species was not possible using morphological characters; thus, it is important to develop an assay for their species‐level identification. Here, the real‐time PCR assays for rapid identification of Z. cucumis and B. jarvisi were developed and validated. The PCR protocols demonstrated their specificity by amplifying the two target species successfully, with no cross‐reactions observed in the tested tephritid species. The in silico test of the primer and probe binding sites of the two assays also demonstrated the assays’ specificity by no mismatches present in the binding regions of the target species, but 1–4 mismatches in the binding regions of the non‐target fruit fly species. The thresholds of detection for the two assays are as low as 10 copies/µl of the target DNA, indicating that the assays have a very high sensitivity. The application of the real‐time PCR assays has greatly assisted in routine pest identifications at the New Zealand border and surveillance programme. Therefore, the assays have the potential to be used by diagnostic agencies and research organizations worldwide.