Study On Rapid Identification Of Quarantine Carpomya Vesuviana Costa Based On Molecular Techniques

At present, Xinjiang jujube cultivation area has more than280,000hm2, becoming the largest fruitgrowing area in Xinjiang. Carpomya vesuviana Costa was found in Turpan region for the first time in2007,which has caused devastating loss to Xinjiang jujube industry and even a spread to other regions. And themost outstanding problem of jujube trading is the identification of quarantine Carpomya vesuviana Costa,both at home and abroad. Therefore, stablishing a method to rapidly identify Carpomya vesuviana Costahas an important meaning.With Carpomya vesuviana Costa as the research object, we determined partial sequences of the mtDNACOI gene and Cytb gene, and then compared the obtained gene fragments with other homologoussequences which have been publicly reported. Application of species-specific primers PCR (SS-PCR) andSYBR Green real-time PCR technology was made to filter out specific primers of Carpomya vesuvianaCosta for rapid identification. The main findings of the research are as follows:1. Genomic DNA of Carpomya vesuviana Costa was extracted firstly. Two different sequences of COIgene and partial sequences of cytb gene of Carpomya vesuviana Costa mtDNA were determined.Carpomya vesuviana Costa of Turpan entered GenBank sequence database for the first time, whose serialnumbers were HQ687210, JX515609and JX101439respectively.2. COI gene sequences (GenBank sequence number: HQ687210) of the mitochondrial DNA (mtDNA)of target Carpomya vesuviana Costa were chosen as the target sequences for the first time. With theCLUSTAL method (DNASTAR, Inc., USA) in Mega4software, the COI gene sequences of Carpomyavesuviana Costa were compared and analyzed with those of other known species of fruit flies (includingsequences in this study and other public report). One pair of Carpomya vesuviana Costa primer wasobtained by screening, which established rapid identification techniques for Carpomya vesuviana Costausing PCR.3. In this study, the new invasion quarantine pest Carpomya vesuviana Costa was rapidly identified,compared to other five kinds of high-frequency fruit flies, using PCR-RFLP technique, which aimed to finda rapid and accurate identification technology for Carpomya vesuviana Costa.4. With the application of self-designed primers and SYBR Green real-time PCR technology, a rapididentification method of Carpomya vesuviana Costa was established. Specific SYBR Green real-time PCRproducts were confirmed by melting curve analysis.5. The mtDNA COI genes, from M7fragment to COOH fragment, of the Quarantine pest Carpomyavesuviana Costa of four different geographic populations were firstly cloned, which initially established aCarpomya vesuviana Costa gene fragment library and provided useful data for molecular biologicalidentification of Carpomya vesuviana Costa.