Recombinant plant dsRNA-binding protein as an effective tool for the isolation of viral replicative form dsRNA and universal detection of RNA viruses
Recombinant plant dsRNA-binding protein as an effective tool for the isolation of viral replicative form dsRNA and universal detection of RNA viruses
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DOI：
10.1007/s10327-009-0155-3
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发表时间：
2009-04-01
影响因子：
1.2
通讯作者：
Sakamoto, Masaru
中科院分区：
文献类型：
--
作者：
Kobayashi, Kappei;Tomita, Reiko;Sakamoto, Masaru
分离病毒复制形式(RF)双链RNA(DsRNA)是一种经典的植物病毒检测技术，当病毒种类不能根据疾病症状预测时。然而，这种方法还没有得到广泛的应用，很可能是因为使用CF-11纤维素提取dsRNA既费力又耗时。在这里，我们报告了另一种工具，重组植物dsRNA结合蛋白，以分离dsRNA。这一工具使我们能够在一小时内从提取的核酸或粗洗涤剂提取物中分离出病毒RF dsRNA。将这项技术与序列非特异性逆转录、聚合酶链式反应扩增、克隆和测序相结合，使用一套试剂和程序就可以有效地检测到各种病毒。
The isolation of viral replicative form (RF) double-stranded RNA (dsRNA) is a classic technique for plant virus detection when the virus species cannot be predicted from disease symptoms. However, the method has not been very widely used, most likely because dsRNA isolation using CF-11 cellulose is laborious and time-consuming. Here we report an alternative tool, a recombinant plant dsRNA-binding protein, to isolate dsRNA. This tool enables us to isolate viral RF dsRNA in an hour from either extracted nucleic acids or crude detergent extracts. Combining this technique with sequence-non-specific reverse transcription, PCR amplification, cloning, and sequencing, a variety of viruses were efficiently detected using a single set of reagents and procedures.