【摘要】 旨在建立适合葡萄的葡萄病毒B的RT-PCR检测技术。从感染葡萄病毒B的葡萄皮层中提取总RNA,以其为模板合成cDNA第一链,并利用两对引物分别进行PCR扩增;回收PCR特异扩增产物,与pUCm-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。结果显示,两对引物均能获得与预期片段大小一致长约460 bp和470 bp的扩增产物扩增片段序列与已报道GVB序列(序列号:X75448)的核苷酸同源性均为81%,经反复多次试验证实,利用总RNA为模板合成cDNA并进行PCR扩增检测GVB是准确、可靠的。更多还原

【Abstract】 The study was to establish a RT-PCR system adaptive to the grapevine virus B(GVB) of grapevine.The total RNA,extracted from grapevine virus B(GVB)-infected Grapevine phloem tissue,was used as template to synthesize the first strain cDNAs.The cDNAs were used for the amplification of GVB by PCR.The PCR products were excised from the gel and purified using the UNIQ-10 Gel Purification kit.The purified RT-PCR products were then cloned into the pUCm-T Vector,and DNA sequencing was carried out by deoxynucleotide chain termination primed with T7 andM13+/-primers from the vector.Result showed that about 460 bp and 470 bp fragments of GVB were obtained based on optimized system.The nucleotide sequences were analysed using the DNAMAN and BLAST software.Compared with GVB isolate reported on GenBank(Accession No.X75448),it showed that both of the isolates obtained here represented 81% identity with it at the sequence level of nucleotide.The study proved that all of these indicated that the PCR method is reliable for detecting GVB.更多还原