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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 virus (SCYLV) -induced sugarcane yellow leaf disease is a new global virus disease. In this paper, the coat protein (CP) gene of Fujian isolate (CHN-FJ1) was cloned by RT-PCR using YLSCPF1 and YLSCPR591 as primers, encoding 196 amino acids. The nucleotide and deduced amino acid sequence of cp gene of SCYLV isolates from different geographical origins were analyzed. The homology was over 95%. According to the conserved sequence of cp gene, a pair of specific primer and TaqMan probe were designed and a real-time TaqMan RT-PCR method of SCYLV was established. The results showed that the detection limit was 1 000 copies / μL (about 3.61 fg / μL) of the original plasmid template DNA, which was 100 times more sensitive than that of the conventional PCR method. Detection of sugarcane mosaic virus, Rhizoctonia solani and pathogen, without typical amplification curve and no Ct value. The detection rate of field sugarcane leaf samples was 100%, 61.5% and 69.2% respectively by real-time fluorescence RT-PCR, conventional RT-PCR and tissue blot immunoblot (TBIA) And TBIA has a higher sensitivity, suitable for the detection of SCYLV.