Establishment And Application Of Gene Chip For Ceratitis Capitata, And Its Related Species And Bactrocera Dorsalis Complex (Diptera: Tephritidae)

Identification of Quarantine fruit fly (Diptera:Tephritidae) is one of the most important technique problem in the international fruits and vegetable trade. The latest published quarantine list of China has includeed the Ceratitis Macleay and Bactrocera Macqurt as detrimental organisms. Current method for identification of fruit fly mainly based on the morphological method. However, it depends on the experience of identifiers and is restrained from the shape and the breeding period of fruit fly. PCR technique is a rapid, precise method for the detection of fruit fly, but current methods based PCR only can detect one sort of fruit fly each time, which can not satisfy the actual applied demands.This study establish a method of genechip for the identifying of Ceratitis capitata, and its related species and the Bactrocera dorsalis complex, according to the difference of sequences of fruit fly, used of genechip technology, by virtue of molecular marker. This technology apply to fruit fly identification, meaning while the genechip have the function of initial selection of Ceratitis Macleay and Bactrocera Macqurt. The study includes three parts:I Preparation the genechip of the Ceratitis capitata, and its related species and the Bactrocera dorsalis complexThe mitochondrial Cytochrome Oxidase I (COI) gene was used as molecular marker, which based on the published data from GenBank. Two pairs of universal primers were designed using DNAstar software to analysis of difference among gene sequence of Anastrepha Schiner, Bactrocera Macqurt, Ceratitis Macleay, Dacus Fabricius and Rhagoletis Loew. The genechip detection system for fruit flies is composed of detecting probes and quality-control probes. The detecting probes include one family probe, three genus probes, two related species probes , six complex species probes and four species specific-probes, and the quality-control probes includeanchor probe, positive probe, negative probe and blank probe. According to designing arrays spotted to substrate, C. capitata, and its related species and the B. dorsalis complex detection chips are parpared.II Optimization of gene chip hybridizationOptimization of hybridization liquid, hybridization time, hybridization temperature. As hybridization temperature is 46℃hybridization time is 3h, hybridization liquid compose of 10×SSPE, 2% SDS, the genechip is specificity and repeatability.III The application of genechip on the C. capitata, and its related species and the B. dorsalis complexCeratitis capitata with different stages and different geographical provenances and its related species (C. rosa and C. cosary) are applied to detect using the prepared genechip.t,. Bactrocera dorsalis complex includes Bactrocera dorsalis, B. papayae, B.carambolae, B. occipitalis, and B. philippinensis are applied to detect using the prepared genechip. The results were exactly same as that of the conventional identification of morphology. Experiments shows genechip technique can be used as rapid detection methods for quarantine fruit fly.