林业科学Issue(4)：60-65,6.DOI:10.11707/j.1001-7488.20140409
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
摘要
Abstract
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.关键词
枣实蝇/SYBR Green/实时荧光 PCR/溶解曲线/鉴定Key words
Carpomya vesuviana/SYBR Green/real-time PCR/melting curve/rapid identification分类
生物科学引用本文复制引用
程晓甜,阿地力·沙塔尔,张伟,李新泉,玛依拉..SYBR Green实时荧光PCR快速鉴定枣实蝇技术[J].林业科学,2014,(4):60-65,6.基金项目
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) （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”。 （）
