实时荧光RT-PCR-步法检测南方菜豆花叶病毒

Detection of Southern bean mosaic virus by one step assay of real-time fluorescent RT-PCR

南方菜豆花叶病毒(Southern bean mosaic virus,SBMV)是我国二类检疫性有害生物,以南方菜豆花叶病毒日本分离物(SBMV-J)总RNA为模板,采用RT-PCR方法扩增病毒外壳蛋白基因及其上游基因的cDNA片断并将其克隆到pMD18-T载体上。序列分析结果表明:SBMV-J cp基因由801个核苷酸组成,编码266个氨基酸,SBMV-J与其它分离物及株系cp基因的核苷酸序列同源性为83%～97%,氨基酸序列同源性为86%～97%。由于SBMV各分离物及株系cp基因的同源性较低,难于设计出较长的普通PCR引物。通过较短引物设计和TaqMan-MGB探针技术,建立了SBMV的实时荧光RT-PCR一步检测方法。该方法的检测低限是0．16 pg,最佳检测总RNA的量是0．16 ng。

Southern bean mosaic virus （SBMV） is in the A2 list concerned to China. The coding region of cp gene from Japan isolate of SBMV was sequenced. It comprised 801 nucleotides and encoded a putative protein of 266 amino acids. The nucleotide and amino acid sequence of cp gene of this isolate shared 83% to 97% and 86% to 97% similarities to those of other SBMV isolates or strains, respectively. A pair of primers and a TaqMan-MGB probe based on the conserved nucleotide sequence of coat protein （CP） gene of SBMV isolates were designed and synthesized. A novel one step assay of real-time fluorescent RT-PCR was established to detect SBMV. The limit content of the detection was 0.16pg and the op- timal content was 0.16 ng.