Real-time qualitative PCR for the inspection and identification of Bactrocera philippinensis and Bactrocera occipitalis (Diptera: Tephritidae) using SYBR Green assay.
The Tephritidae fruit fly pests, Bactrocera philippinensis (Drew and Hancock) and B. occipitalis (Bezzi), are two sympatric species of the 52 B. dorsalis complex with significant quarantine importance in Southeast Asia. A 1356 base pairs of DNA fragment from the mitochondrial Cytochrome Oxidase I (COI) genes of B. philippinensis and B. occipitalis was sequenced and analysed, respectively. The similarity of COI gene between these two species is 96.8%. Two sets of species-specific primers were designed for polymerase chain reaction (PCR). A technique of the combination of real-time qualitative PCR using SYBR Green assay with melting curve analysis is developed to detect and identify these two Bactrocera sympatric species. This assay exhibited high specificity, reliability, as well as its high speed, low cost and the possibility of automating. Thus SYBR Green real-time PCR can be used as a rapid and specific technique in species identification of quarantine pests.