SYBR Green实时荧光PCR快速鉴定枣实蝇技术  

Rapid Identification of Carpomya vesuviana by Real-Time PCR with SYBR Green Chemical

作　　者：程晓甜[1] 阿地力·沙塔尔[1] 张伟[2] 李新泉[1] 玛依拉[1] 

机构地区：[1]新疆农业大学林学与园艺学院、新疆教育厅干旱区林业生态与产业技术重点实验室,乌鲁木齐830052 [2]新疆出入境检验检疫局技术中心,乌鲁木齐830083

出　　处：《林业科学》2014年第4期60-65,共6页Scientia Silvae Sinicae

基　　金：新疆维吾尔自治区科技计划项目“特色林果重大病虫持续高效绿色防控技术研究”(201130102-3);新疆维吾尔自治区自治区重点学科森林培育资助项目;新疆维吾尔自治区林业有害生物防治检疫局项目“枣实蝇快速检测和监测技术研发”

摘　　要：应用SYBR Green实时荧光PCR技术,建立SYBR Green实时荧光PCR快速鉴定枣实蝇的方法。利用枣实蝇（mtDNA）中COⅠ基因序列,设计筛选出1对种的特异引物CarF/CarR。引物的特异性分别用桔小实蝇、瓜实蝇、南瓜实蝇、番石榴实蝇和桃果实蝇5种实蝇来验证。SYBR Green PCR实时荧光反应的灵敏度用40,20,10,1,0.1,0.01,0.001 ng·μL^-1 7个浓度枣实蝇DNA模板来检测。结果表明：SYBR Green PCR的检测限度达0.01 ng·μL^-1以下,最适模板DNA浓度为1～20 ng·μL^-1。SYBR Green实时荧光PCR的可靠性可用枣实蝇不同虫态（幼虫、蛹、成虫）的扩增曲线、熔解曲线及PCR产物的琼脂糖凝胶电泳来检验。成虫、幼虫和蛹3种不同虫态的枣实蝇有一致的扩增曲线,熔解曲线分析和琼脂糖凝胶电泳条带分别用来确认实时荧光PCR产物的特异性;其平均熔解温度为（75.3±0.1）℃,并获得一段长为205 bp的特异性目标片段,说明在枣实蝇成虫为模板的基础上建立的SYBR Green实时荧光PCR方法同时适用于幼虫、蛹的快速鉴定,可将不同虫态的枣实蝇与近似种类区分开来。The technique of a real-time PCR with SYBR Green I dye was used in this study. A SYBR Green real-time PCR was developed to rapidly obtained identify Carpomya vesuviana BIOER FQD- 48A sequence detection system. A C. vesuviana specific PCR primers set was designed based on mtDNA COⅠ gene of C. vesuviana. Five Bactrocera fruit flies,B. dorsalis,B. cucurbitae,B. tau,B. correcta and B. zonata,were used to determine the specificity of the primers set CarF / CarR. A series of genomic DNA dilutions of C. vesuviana,40,20,10,1,0. 1,0. 01,0. 001 ng·μL^- 1,were used to detect the sensitivity of SYBR Green PCR. The results showed that the detection limit of SS-PCR was less than 0. 01 ng ·μL^- 1. The template DNA concentration was one of the sources of variability in cycle threshold values（ CT） and the optimum DNA concentration was between 1 ng·μL^- 1and 20 ng·μL^- 1in SYBR Green PCR. The template DNA isolated from the larva,pupa and adult specimens of C. vesuviana respectively were used to detect the reliability of SYBR Green PCR. Melting curve analysis（ MCA） and agarose gel electrophoresis（ AGE） was then used to confirm the specificity reliability of PCR products,respectively. The similar amplification plots were obtained in three different stages of C. vesuviana. The average melting temperature（ Tm） of the PCR product from B. latifrons was 75. 3 ℃ ± 0. 1 ℃,and a 205 bp length fragment target was amplified. Except for the adult of fruit flies,the molecular techniques established in this study can also rapidly identify the larva and pupa.

关 键 词：枣实蝇 SYBR Green 实时荧光PCR 溶解曲线 鉴定 

分 类 号：Q965[生物学—昆虫学]