为了建立葫芦科作物干种子主要种传病毒检测方法,在现有的TRIZOL法、CTAB法等提取植物总RNA方法的基础上进行改良,从富含有多糖的甜瓜干种子中提取高质量的总RNA。建立了18 S r RNA为内参基因的侵染葫芦科作物4种主要种传病毒的两重RT-PCR检测方法。应用改良TRIZOL法分别提取单粒和0.1 g(约20粒)带毒甜瓜种子的总RNA作为检测模板,分别对4种种传病毒进行RT-PCR检测,结果表明可分别从携带4种种传病毒的甜瓜种子上检测到对应的病毒,18 S r RNA基因能够很好的作为内参基因对病毒检测结果进行评估。
Imported cowpea seeds were detected with growing test,ELISA assay and RT-PCR method.The ELISA results showed that cowpea seedlings with symptoms reacted positively with antibody against Southern bean mosaic virus(SBMV).The 979 bp of fragment could be amplified from two positive ELISA samples using primers specific for Southern cowpea mosaic virus(SCPMV),and the sequence determination results proved that the pathogen existing in imported cowpea seeds was SCPMV.The positive ELISA results with SBMV antibody could be further confirmed by RT-PCR amplification with specific primers designed to amplify the coat protein gene and 3’ noncoding region of SCPMV and SBMV.The RT-PCR method presented here was suitable for molecular identification of SBMV and SCPMV in entry-exit plant quarantine laboratories.
