Rapid Identification Of 5 Important Quarantine Fruit Flies By Species-specific Primer COI(SS-COI)

Many fruit flies are pests for fruits and vegetables.With the globalization of world economy,trades between countries are increasingly frequent.Fruit flies cause more and more attention in the international trade of fruits and vegetables due to their diffusion invasion.The traditional identification method is based on morphological characteristics of adults,but fruit flies intercepted at port sites were often immature stages,they usually needed to be reared until adults presented in order to identify them.The customs clearance speed of fruit and vegetable trade has been seriously affected by the factor of time,stages,experience and so on,thus mastering an important harmful fruit fly especially rapid identification of immature stages has important significance in quarantine at ports.Five pairs of species-specific primers,JF190 and JR386,NF404 and NR610,GF85 and GR531,TF37 and TR480,FL680 and RL1057 were designed based on mitochondria COI sequences from NCBI.Softwares such as Bioedit,Primer-Premier 5,Oligo 6.44 were used.They could be accurately identified with the Bactrocera scutellata(Hendel),Bactrocera tau(Walker),Bactrocera cucurbitae(Coquillett),Bactrocera atrifemur Drew&Hancock,Bactrocera latifrons(Hendel)respectively.The selection of target fruit fly as a positive control,and other 16 species of fruit flies as a negative control,were amplified by PCR and the PCR products were electrophoresis.The results show,only the target of fruit fly could be amplified bands that are clear and single,which is 197 bp,207 bp,447 bp,444 bp,378 bp,the remaining fruit flies having no bands.The use of nucleic acid and protein analyzer to test the extraction of target fruit fly DNA template concentration,and the target fruit fly DNA template is based on a dilution gradient by 10-1,10-2,10-3 times,then different concentrations of DNA template are taken out for the lowest detection threshold determination.The B.scutellata,B.tau,,B.cucurbitae,B.atrifemur,B.latifrons,their lowest detection threshold were 2.889×10-2 ng/?L,5.695×10-1 ng/?L,2.162 ng/?L,4.517×10-1 ng/?L,25.21 ×10-1 ng/?L respectively.The SS-PCR identification method has been put to quarantine work,showing that the method is of high specificity,and can be used in the epidemic monitoring and port quarantine inspection of fruit fly.Using species-specific COI primers can rapidly identify the five fruit fly species at different development stages within 8 h.It has the advantage of simple and fast,strong specificity,and high sensitivity,and can meet the requirements of the port inspection and quarantine clearance.