Varanda C M R, Cardoso J M S, Oliveira M D M, Oliveira S, Clara M I E, Félix M R F
Laboratório de Virologia Vegetal, Instituto de Ciências Agrárias e Ambientais Mediterrânicas, Universidade de Évora, Apartado 94, 7002-554 Évora, Portugal.
IMAR-CMA, Departamento de Ciências da Vida, Universidade de Coimbra, 3004-517 Coimbra, Portugal.
J Virol Methods. 2014 Nov;208:63-5. doi: 10.1016/j.jviromet.2014.08.004. Epub 2014 Aug 10.
The high infection levels due to Olive latent virus 1 (OLV-1), Olive mild mosaic virus (OMMV) (alphanecrovirus) and Tobacco necrosis virus D (TNV-D) (betanecrovirus) in Portuguese olive orchards prompted us to develop a rapid PCR-based assay for the simultaneous detection of these viruses aimed at the sanitary selection and marketing of plant material in compliance with European Union regulations. A pair of degenerate oligonucleotide primers, parRdRp5' and parCoat3' was designed based on conserved regions located in the RNA-dependent RNA polymerase (RdRp) and coat protein (CP) genes of these viruses and one other alphanecrovirus, Tobacco necrosis virus A. Its use in RT-PCR assays generated a product of ca. 2000 bp for the 4 viral species tested. These primers were compared with virus specific primers in multiplex RT-PCR, and identical results were obtained. Its application to dsRNA extracted from 54 olive field growing trees originated the expected ca. 2000 bp amplicon in 17 trees. The virus identity was determined by sequencing the cloned RT-PCR products. No TNV-A was found. The RT-PCR assay using the degenerate primers described in this study were shown to be reliable in detecting any of the above-mentioned alpha- and betanecroviruses, and it is as sensitive as that which uses virus specific primers in multiplex assays. Therefore, this assay is well suited for the rapid screen of virus-free plant material in selection and improvement crop programmes. Additionally, it has the potential to reveal virus diversity and the presence of new viruses, provided the RT-PCR generated amplicon is further sequenced.
葡萄牙橄榄园中因感染油橄榄潜伏病毒1（OLV-1）、油橄榄轻度花叶病毒（OMMV）（α坏死病毒属）和烟草坏死病毒D（TNV-D）（β坏死病毒属）而导致的高感染水平促使我们开发一种基于PCR的快速检测方法，用于同时检测这些病毒，以符合欧盟法规对植物材料进行卫生筛选和销售。基于这些病毒以及另一种α坏死病毒属病毒烟草坏死病毒A的RNA依赖RNA聚合酶（RdRp）和外壳蛋白（CP）基因中的保守区域，设计了一对简并寡核苷酸引物parRdRp5'和parCoat3'。在RT-PCR检测中，对于所测试的4种病毒，使用该引物产生了约2000 bp的产物。在多重RT-PCR中，将这些引物与病毒特异性引物进行了比较，得到了相同的结果。将其应用于从54株田间生长的橄榄树中提取的dsRNA，在17株树中产生了预期的约2000 bp扩增子。通过对克隆的RT-PCR产物进行测序来确定病毒的身份。未发现烟草坏死病毒A。本研究中描述的使用简并引物的RT-PCR检测方法在检测上述任何一种α和β坏死病毒时都被证明是可靠的，并且在多重检测中与使用病毒特异性引物的检测方法一样灵敏。因此，该检测方法非常适合在作物选择和改良计划中快速筛选无病毒植物材料。此外，只要对RT-PCR产生的扩增子进一步测序，它就有可能揭示病毒的多样性和新病毒的存在。
