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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) was the second quarantine pest in China. The total RNA of SBMV-J was used as a template to amplify the virus shell by RT-PCR The cDNA fragment of the protein gene and its upstream gene was cloned into the pMD18-T vector. Sequence analysis showed that the SBMV-J cp gene consisted of 801 nucleotides and encoded 266 amino acids. The nucleotide sequence of SBMV-J shared 83% -97% identity with other isolates and strains of cp gene. The amino acid sequence homology is 86% ~ 97%. Due to the low homology of cp genes between isolates and strains of SBMV, it is difficult to design long PCR primers. A real-time fluorescent RT-PCR one-step method was established for SBMV by using the short primer design and TaqMan-MGB probe technology. The detection limit of this method is 0.16 pg, and the optimal amount of total RNA detected is 0.16 ng.