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 -17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。
Rapid Identification of Carpomya vesuviana by Real-Time PCR with SYBR Green Chemical
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 -1 and 20 ng·μL -1 in 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.
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维普
