甘蔗黄叶病毒外壳蛋白基因克隆及其实时荧光RT-PCR检测

 甘蔗黄叶病毒（Sugarcane yellow leaf virus, SCYLV）引起的甘蔗黄叶病是一种新的全球性病毒病害。本文以YLSCPF1和YLSCPR591为引物，采用RT-PCR方法克隆了甘蔗黄叶病毒福建分离物（CHN-FJ1）外壳蛋白（CP）基因，编码196个氨基酸。分析不同地理来源的SCYLV病毒分离物cp基因核苷酸及其推导编码的氨基酸序列，同源性达95%以上。根据cp基因的保守序列，设计1对特异性引物和TaqMan探针，建立了SCYLV的TaqMan实时荧光RT-PCR方法。结果表明，检测下限为初始质粒模板DNA 1 000拷贝/μL（约3.61 fg/μL），比常规PCR方法的灵敏度提高100倍。检测甘蔗花叶病毒、宿根矮化病菌和黑穗病菌，没有典型的扩增曲线和无Ct值。应用实时荧光RT-PCR、常规RT-PCR和组织印迹免疫杂交（TBIA）对田间甘蔗叶片样品进行检测，阳性检出率分别为100%、61.5%和69.2%，表明该方法比常规RT-PCR和TBIA具有更高的灵敏度，适合于对SCYLV的检测。

Cloning and detection of coat protein gene of Sugarcane yellow leaf virus by real-time fluorescent RT-PCR

Sugarcane yellow leaf disease induced by Sugarcane yellow leaf virus(SCYLV) is a global viral disease.The coat protein gene(cp) that deduced 196 amino acids from CHN-FJ1 isolate was cloned by RT-PCR with the primers YLSCPF1and YLSCPR591.Sequence analysis showed that identity of nucleotides and deduced amino acids shared above 95% among the isolates from different geographical origins.The primers and TaqMan probe located on the conservative sequence of cp were designed for real-time fluorescent RT-PCR.The st...