Use of degenerate primers for partial sequencing and RT-PCR-based assays of grapevine leafroll-associated viruses 4 and 5
Use of degenerate primers for partial sequencing and RT-PCR-based assays of grapevine leafroll-associated viruses 4 and 5
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DOI：
10.1094/phyto.1998.88.11.1238
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发表时间：
1998-11-01
期刊：
影响因子：
3.2
通讯作者：
Rowhani, A
中科院分区：
文献类型：
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作者：
Routh, G;Zhang, YP;Rowhani, A
从感染葡萄藤叶相关病毒4（GLRaV-4）和5（GLRaV-5）的葡萄中纯化双链RNA（dsRNA）。使用简并寡核苷酸对该dsRNA进行逆转录酶聚合酶链反应（RT-PCR），所述简并寡核苷酸被设计为从已知的环状病毒的热休克蛋白70同源物（HSP 70）扩增约550至650个核苷酸的片段。凝胶分离适当分子量的RT-PCR产物并克隆到质粒载体pGEM-T中。对通过使用这些引物对从感染GLRaV-4的植物分离的dsRNA产生的RT-PCR产物的克隆进行测序。该序列用于开发能够检测GLRaV-4的免疫捕获RT-PCR（IC-RT-PCR）检测方案。从分离自感染GLRaV-5的植物的dsRNA制备类似的克隆。还对这些克隆进行了测序。比较了这两个序列，并开发了能够从两者扩增cDNA的RT-PCR引物。这些实验证明，扩增梭状病毒HSP 70序列的简并引物可用于成功地产生可用于这些病毒的IC-RT-PCR检测的序列。这些数据也表明，它是可行的，使用HSP 70序列设计的PCR引物能够更普遍的PCR检测多种GLRaV血清型。最后，在这些推定的梭状病毒中存在梭状病毒样HSP 70序列意味着它们确实是该分类组的成员。
Double-stranded RNA (dsRNA) was purified from grapevines infected with grapevine leafroll-associated viruses 4 (GLRaV-4) and 5 (GLRaV-5), two putative closteroviruses. Reverse-transcriptase polymerase chain reaction (RT-PCR) was performed on this dsRNA using degenerate oligonucleotides designed to amplify an approximately 550- to 650-nucleotide fragment from the heat shock protein 70 homolog (HSP70) of the known closteroviruses. RT-PCR products of the appropriate molecular weight were gel-isolated and cloned into the plasmid vector pGEM-T. Clones of RT-PCR products generated by using these primers on dsRNA isolated from a plant infected with GLRaV-4 were sequenced. This sequence was used to develop an immunocapture RT-PCR (IC-RT-PCR) detection protocol capable of detecting GLRaV-4. Similar clones were made from dsRNA isolated from a plant infected with GLRaV-5. These clones were also sequenced. The two sequences were compared, and RT-PCR primers were developed that were able to amplify cDNA from both. These experiments demonstrate that degenerate primers that amplify closterovirus HSP70 sequences can be used to successfully generate sequences useful for IC-RT-PCR detection of these viruses. These data also suggest that it is feasible to use HSP70 sequences to design PCR primers capable of more general PCR detection of multiple GLRaV serotypes. Lastly, the presence of closterovirus-like HSP70 sequences in these putative closteroviruses implies that they are indeed members of this taxonomic group.