甘蔗黄叶病毒外壳蛋白基因克隆及其实时荧光RT-PCR检测

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

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。

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 studies demonstrated that the detection limit was 1 000 copies/μL（3.61 fg/μL） of positive cloning plasmid,was 100-fold higher than that of regular RT-PCR.Typical amplification curve and Ct value did not appear in detection of Sugarcane mosaic virus（SCMV）,the pathogens of sugarcane ratoon stunning di-sease（Leifsonia xyli subsp.xyli） and sugarcane smut（Ustilago scitaminea Syd.）.The detection of leaf samples from field were conducted by real-time fluorescent RT-PCR,regular RT-PCR as well as tissue blot immunoassay（TBIA） and positive results reached 100%,61.5% and 69.2%,respectively.The real-time fluorescent RT-PCR was more sensitive and reliable method for detection of SCYLV as compared with regular RT-PCR and TBIA.