Combined DECS Analysis and Next-Generation Sequencing Enable Efficient Detection of Novel Plant RNA Viruses.
Combined DECS Analysis and Next-Generation Sequencing Enable Efficient Detection of Novel Plant RNA Viruses.
复制标题
DOI：
10.3390/v8030070
复制
发表时间：
2016-03-07
期刊：
影响因子：
--
通讯作者：
Sekine KT
中科院分区：
文献类型：
--
作者：
Yanagisawa H;Tomita R;Katsu K;Uehara T;Atsumi G;Tateda C;Kobayashi K;Sekine KT
植物细胞内高分子量双链RNA（dsRNA）的存在是RNA病毒感染的指标，因为这些病毒具有基因组或复制型dsRNA。德克斯（dsRNA分离、穷举扩增、克隆和测序）分析已被证明能够检测未知病毒。我们假设德克斯分析和下一代测序（NGS）的组合将提高该技术的检测效率和可用性。在这里，我们描述了一个模型的情况下，我们有效地检测到推定的基因组序列的蓝莓鞋带病毒（BSSV），属Sobemovirus的成员，这到目前为止还没有报道。使用dsRNA结合蛋白从BSSV感染的蓝莓植物中分离dsRNA，逆转录，扩增，并使用NGS测序。获得了与来自其他Sobemovirus物种的序列具有相似性的4，020个核苷酸（nt）的重叠群作为BSSV基因组序列的候选者。基于该重叠群序列的逆转录（RT）-PCR引物组能够在所有受BSSV感染的植物中检测到BSSV，但在健康对照中检测不到。由推定的外壳蛋白基因编码的重组蛋白与BSSV抗体结合，表明候选序列是BSSV本身的序列。我们的研究结果表明，德克斯分析和NGS的组合，在这里指定为“DECS-C”，是一个强大的方法检测新的植物病毒。
The presence of high molecular weight double-stranded RNA (dsRNA) within plant cells is an indicator of infection with RNA viruses as these possess genomic or replicative dsRNA. DECS (dsRNA isolation, exhaustive amplification, cloning, and sequencing) analysis has been shown to be capable of detecting unknown viruses. We postulated that a combination of DECS analysis and next-generation sequencing (NGS) would improve detection efficiency and usability of the technique. Here, we describe a model case in which we efficiently detected the presumed genome sequence of Blueberry shoestring virus (BSSV), a member of the genus Sobemovirus, which has not so far been reported. dsRNAs were isolated from BSSV-infected blueberry plants using the dsRNA-binding protein, reverse-transcribed, amplified, and sequenced using NGS. A contig of 4,020 nucleotides (nt) that shared similarities with sequences from other Sobemovirus species was obtained as a candidate of the BSSV genomic sequence. Reverse transcription (RT)-PCR primer sets based on sequences from this contig enabled the detection of BSSV in all BSSV-infected plants tested but not in healthy controls. A recombinant protein encoded by the putative coat protein gene was bound by the BSSV-antibody, indicating that the candidate sequence was that of BSSV itself. Our results suggest that a combination of DECS analysis and NGS, designated here as “DECS-C,” is a powerful method for detecting novel plant viruses.