SYBR Green实时荧光PCR快速鉴定辣椒实蝇

Rapid identification of Bactrocera latifrons by real-time PCR using SYBR Green chemistry

本文应用SYBRGreen实时荧光PCR技术,建立了SYBRGreen实时荧光PCR快速鉴定辣椒实蝇的方法。利用辣椒实蝇mtDNACOI基因序列,筛选出种特异引物lati1/lati2。引物的特异性分别用辣椒实蝇、橘小实蝇、木瓜实蝇、杨桃实蝇、菲律宾实蝇、芒果实蝇、番石榴实蝇、瓜实蝇和南瓜实蝇等9种果实蝇来验证。0.01ng,0.1ng,1ng,10ng,20ng,40ng和100ng等7个不同浓度系列的辣椒实蝇DNA模板用来检测SYBRGreenPCR实时荧光反应的灵敏度,结果表明,SYBRGreenPCR的检测限度达1pg以下,最适模板DNA浓度为1～20ng。成虫、幼虫和蛹等3种不同虫态的辣椒实蝇有一致的扩增曲线,熔解曲线分析和琼脂糖凝胶电泳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77.5℃±0.1℃。获得1段长为366bp的特异性目标片段,说明在辣椒实蝇成虫为模板基础上建立的SYBRGreen实时荧光PCR方法适于幼虫、蛹的快速鉴定。

The technique of a real-time PCR using SYBR Green Ⅰ dye was introduced in this study. We had developed a SYBR Green real-time PCR to rapid identify Bactrocera haifrons on ABI PRISM 7700 sequence detection system. A lati- fron-specific PCR primers set was obtained based on mtDNA COI gene of B. latifrons. Nine Bactrocera fruit flies, B. latifrons, B dorsalis, B. papayae, B. carambolae, B. philippinensis, B. occipitalis, B. correcta, B. cucurbitae and B. tau, were used to determine the specificity of primers set lati1/lati2. A series of genomic DNA dilution of B. latifrom, 0.01ng, 0.1ng, 1ng, 10ng, 20ng, 40ng and 100ng, were used to detect the sensitivity of SYBR Green PCR. The template DNA concentration was one of the sources of variability in cycle threshold values （CT） and the optimum DNA concentration was between lng and 20ng in SYBR Green PCR. The template DNA isolated from the larva, pupa and adult specimens of B. latifrom respectively were used to detect the reliability of SYBR Green PCR. Melting curve analysis （MCA） and agarose gel electrophoresis （AGE） was followed to confirm the specificity reliability of PCR prod- ucts, respectively. The results showed the similar amplification plots were gotten in three different stages of B. latifrons. The average melting temperature （Tm） of the PCR product from B. latifrons was 77.5℃± 0.1℃. And a 366bp length fragment target was amplified respectively.