Department of Microbiology and Plant Pathology, University of Pretoria, Pretoria, 0002, South Africa; Forestry and Agricultural Biotechnology Institute, University of Pretoria, Pretoria, 0002, South Africa.
Department of Microbiology and Plant Pathology, University of Pretoria, Pretoria, 0002, South Africa; Forestry and Agricultural Biotechnology Institute, University of Pretoria, Pretoria, 0002, South Africa; Genetics Department, University of Stellenbosch, Stellenbosch, 7600, South Africa.
J Virol Methods. 2018 Sep;259:39-44. doi: 10.1016/j.jviromet.2018.05.014. Epub 2018 May 31.
Soybean blotchy mosaic virus (SbBMV), a plant virus of the genus Cytorhabdovirus is an economically important virus of soybean reported only from the warmer, lower-lying soybean production areas in South Africa. The virus consistently appears in soybean crops annually in spite of the absence of soybean plants in winter. One possible reason for this may be that the virus replicates and hence persists in the SbBMV vector, a leafhopper, Peragallia caboverdensis. RNA viruses with antisense genomes as inferred for SbBMV produce positive sense RNAs as intermediate replicative forms during replication in their hosts, and detection of the positive strand in the plant host or vector is evidence of virus replication. In this study, a positive-strand specific RT-PCR (pss-RT-PCR) was developed to detect the positive RNA strand of SbBMV and validated on nine SbBMV isolates from soybean. The effect of tagged reverse transcription (RT) primers for cDNA synthesis, coupled with PCR using a tag-specific primer, as well as removal of unincorporated RT primers following cDNA synthesis was assessed. The positive RNA strand of SbBMV in infected plants was successfully detected following this protocol. Reverse transcription with forward and unmodified reverse primers confirmed that the assay was not able to detect the genomic sense RNA or self-primed cDNAs, lacking the non-viral tag, respectively. However, Exonuclease I (ExoI) treatment of cDNA was required to eliminate false-positive results during PCR amplification.
大豆斑纹花叶病毒（SbBMV）是一种植物呼肠孤病毒属病毒，是南非较温暖、地势较低的大豆产区报告的一种具有经济重要性的大豆病毒。尽管冬季没有大豆植株，但该病毒每年都会在大豆作物中出现。造成这种情况的一个可能原因是，病毒在其载体叶蝉 Peragallia caboverdensis 中复制并因此得以持续存在。具有反义基因组的 RNA 病毒在宿主中复制时会产生正链 RNA 作为中间复制形式，并且在植物宿主或载体中检测到正链就证明了病毒的复制。在本研究中，开发了一种用于检测 SbBMV 正链的正链特异性 RT-PCR（pss-RT-PCR），并在来自大豆的 9 个 SbBMV 分离株上进行了验证。评估了标记反转录 (RT) 引物对 cDNA 合成的影响，以及与使用标记特异性引物的 PCR 结合，以及 cDNA 合成后未掺入的 RT 引物的去除。按照该方案成功检测到感染植物中的 SbBMV 正链。使用正向和未修饰的反向引物进行反转录证实，该测定法无法分别检测到基因组感测 RNA 或缺乏非病毒标记的自我启动 cDNA。然而，在 PCR 扩增过程中需要 Exonuclease I (ExoI) 处理 cDNA 以消除假阳性结果。
