草莓镶脉病毒的PCR检测及特异片段的序列分析

Detection of strawberry vein banding virus by PCR and sequence analysis of specific fragment

用CTAB法从感病的草莓叶片中提取总DNA,以其为模板经PCR扩增获得与预期片段大小一致长约600bp的扩增产物,同时优化PCR反应程序,获得单一特异条带;通过总DNA浓度梯度稀释,进行PCR扩增,结果表明能检测到2.5μg叶组织中病毒的存在。回收PCR特异扩增产物,与pMD18-T载体连接,并进行转化、重组克隆的筛选、重组质粒的酶切鉴定和序列测定。扩增片段序列与已报道SVBVCP基因序列(序列号:Nc_001725)的核苷酸同源性为89.2%,氨基酸同源性为96.3%。该特异片段序列在GenBank中的登记号为AY862389。

The total DNA, extracted by using CTAB method from SVBV-infected strawberry leaves, was used as template to amplify by PCR. PCR amplification conditions and programs were optimized. About 600 bp fragment of SVBV was obtained based on optimum system. The total DNA was diluted to density gradient and amplified by PCR. The results showed that it could detect the virus in 2.5 μg leaf tissue. PCR method is a reliable method for detecting SVBV. The PCR specific fragment was recycled, cloned into pMD18-T vector and sequenced. Compared with SVBV isolate that reported (GenBank accession number Nc_001725), it showed that the sequence of coat protein gene of this isolate was 89.2% and 96.3% identity at the sequence levels of nucleotide and deduced amino acid. GenBank accession number of the specific fragment sequence was AY862389.