摘要
采用T-A克隆技术,在白纹伊蚊β-肌动蛋白的基因序列上设计引物,PCR扩增后将特异性产物连入T载体中,提取质粒,经测序分析验证后,测定浓度,稀释获得标准品,采用SYBR green法进行实时定量PCR,并绘制标准曲线,作为白纹伊蚊各种基因实时定量PCR检测中的内参照物.结果表明：利用此标准品制备的标准曲线具有较高的扩增效率和良好的线性关系（斜率为-3.3287,R2=0.9980）;实时定量PCR熔解曲线分析表明,温度在83℃±0.5℃的PCR产物是白纹伊蚊肌动蛋白序列上的特异性产物,表明此标准品是白纹伊蚊肌动蛋白特异性的.本实验成功建立白纹伊蚊β-actin基因实时定量PCR方法,可作为内参照物运用于白纹伊蚊基因差异表达的研究.
Primers were designed based on the Aedes albopictus [3-actin gene, the fragment of Ae. albopictus β-actin gene was amplified from the cDNA and linked with T vector. The DNA was confirmed by sequencing and was diluted as standard for quantitative real-time polymerase chain reaction assay of SYBR green method. The results showed a good linear negative regression between threshold cycle （Ct） and Log starting quantity of copy number. The melting curve of real-time PCR showed melting temperature at 83℃ ＋ 0. 5℃, indicating PCR products were that of the β-actin gene sequence and thus the standard used in this study was specific for β-actin gene. Real-time quantitative PCR for β-actin analysis is established successfully.
出处
《寄生虫与医学昆虫学报》
CAS
2010年第4期224-227,共4页
Acta Parasitologica et Medica Entomologica Sinica
基金
国家自然科学基金项目（No.30700691）
