A real‐time PCR toolbox for accurate identification of invasive fruit fly species

Abstract

Significant plant pests such as fruit flies that travel with fresh produce between countries as eggs or larvae pose a great economic threat to the agriculture and fruit industry worldwide. Time‐limited and expensive quarantine decisions require accurate identification of such pests. Immature stages are often impossible to identify, making them a serious concern for biosecurity agencies. Use of COI barcoding PCR, often the only molecular identification resource, is time‐consuming. We assess the suitability of the COI barcoding region for real‐time PCR assays to identify four pest fruit fly species (Family: Tephritidae), in a diagnostic framework. These species, namely Mediterranean fruit fly (Ceratitis capitata), Queensland fruit fly (Bactrocera tryoni), African invader fly (Bactrocera invadens) and Island fly (Dirioxa pornia) each provide a different set of genetic species delimitation problems. We discuss the benefits and limitations of using a single‐gene TaqMan™ real‐time approach for such species. Our results indicate that COI‐based TaqMan™ real‐time PCR assays, in particular for genetically distinct species, provide an accurate, sensitive and rapid diagnostic tool.

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

“…The annealing temperature of 65°C was selected for the real‐time PCR assays for B. tryoni complex due to the cross‐reaction with the closely related species (Dhami et al, ); however, at this temperature (65°C), lower amplification efficiency was observed for the real‐time PCR assays of Z. cucumis or B. jarvisi in the current study. On the other hand, the assays for Z. cucumis or B. jarvisi have the possibilities to be used as multiplex with the assays targeting other Tephritidae interceptions, such as B. dorsalis complex, Dirioxa pornia and Ceratitis capitata (Dhami et al, ). Further validations are required prior to application of these as multiplex assays in diagnostics.…”

“…The real‐time PCR assays developed in this study have been validated in one tube as duplex assay if the intercepted Tephritidae samples are suspected to be either Z. cucumis or B. jarvisi . For the current assay formats, it is not possible to multiplex the real‐time PCR assays for Z. cucumis or B. jarvisi in this study with the real‐time PCR assay for B. tryoni complex (Dhami et al, ) due to the difference in the annealing temperatures. The annealing temperature of 65°C was selected for the real‐time PCR assays for B. tryoni complex due to the cross‐reaction with the closely related species (Dhami et al, ); however, at this temperature (65°C), lower amplification efficiency was observed for the real‐time PCR assays of Z. cucumis or B. jarvisi in the current study.…”

“…For the current assay formats, it is not possible to multiplex the real‐time PCR assays for Z. cucumis or B. jarvisi in this study with the real‐time PCR assay for B. tryoni complex (Dhami et al, ) due to the difference in the annealing temperatures. The annealing temperature of 65°C was selected for the real‐time PCR assays for B. tryoni complex due to the cross‐reaction with the closely related species (Dhami et al, ); however, at this temperature (65°C), lower amplification efficiency was observed for the real‐time PCR assays of Z. cucumis or B. jarvisi in the current study. On the other hand, the assays for Z. cucumis or B. jarvisi have the possibilities to be used as multiplex with the assays targeting other Tephritidae interceptions, such as B. dorsalis complex, Dirioxa pornia and Ceratitis capitata (Dhami et al, ).…”

“…Real‐time PCR assay for B. tryoni complex and other fruit fly species has been developed and routinely used for diagnostics in New Zealand (Dhami et al, ; Li, Waite, Gunawardana, et al, ), but no real‐time PCR assay for Z. cucumis and B. jarvisi has been reported. Prior to the development of the real‐time PCR assays, if the suspected Tephritidae interceptions were tested negative to B. tryoni complex (Dhami et al, ), the sample needed to be barcoded for the identification to species. Given the biosecurity implications, rapid identification techniques are much warranted for any intercepted and detected fruit fly species.…”

“…(MacKenzie, Vincent, Burgher‐MacLellan, & Gaul, ), Bactrocera latifrons , Bactrocera philippinensis (current Bactrocera dorsalis ) and Bactrocera occipitalis (MacKenzie et al, ; Yu, Chen, Zhang, & Yin, ; Yu, Zhang, Chen, Zhang, & Yin, ). Several real‐time PCR assays developed for fruit flies are used in the routine diagnostics at the New Zealand borders, including B. tryoni complex , B. dorsalis complex , Dirioxa pornia, Ceratitis capitata (Dhami, Gunawardana, Voice, & Kumarasinghe, ) and Bactrocera xanthodes complex ( B. xanthodes, B. neoxanthodes and B. paraxanthodes ) (Li, Waite, Gunawardana, et al, ) and those real‐time PCR assays could provide rapid identification in 4 hr. However, no real‐time PCR protocols for Z. cucumis and B. jarvisi have been developed yet.…”