Treder Krzysztof, Chołuj Joanna, Zacharzewska Bogumiła, Babujee Lavanya, Mielczarek Mateusz, Burzyński Adam, Rakotondrafara Aurélie M
Laboratory of Molecular Diagnostic and Biochemistry, Bonin Research Center, Plant Breeding and Acclimatization Institute-National Research Institute, 76-009, Bonin, Poland.
Department of Plant Pathology, University of Wisconsin-Madison, 1630 Linden Drive, Madison, WI, 53706, USA.
Arch Virol. 2018 Feb;163(2):447-458. doi: 10.1007/s00705-017-3635-3. Epub 2017 Nov 8.
Potato virus Y (PVY) infection has been a global challenge for potato production and the leading cause of downgrading and rejection of seed crops for certification. Accurate and timely diagnosis is a key for effective disease control. Here, we have optimized a reverse transcription loop-mediated amplification (RT-LAMP) assay to differentiate the PVY O and N serotypes. The RT-LAMP assay is based on isothermal autocyclic strand displacement during DNA synthesis. The high specificity of this method relies heavily on the primer sets designed for the amplification of the targeted regions. We designed specific primer sets targeting a region within the coat protein gene that contains nucleotide signatures typical for O and N coat protein types, and these primers differ in their annealing temperature. Combining this assay with total RNA extraction by magnetic capture, we have established a highly sensitive, simplified and shortened RT-LAMP procedure as an alternative to conventional nucleic acid assays for diagnosis. This optimized procedure for virus detection may be used as a preliminary test for identifying the viral serotype prior to investing time and effort in multiplex RT-PCR tests when a specific strain is needed.
马铃薯Y病毒（PVY）感染一直是全球马铃薯生产面临的挑战，也是种薯作物降级和认证不合格的主要原因。准确及时的诊断是有效控制病害的关键。在此，我们优化了一种逆转录环介导等温扩增（RT-LAMP）检测方法，以区分PVY的O和N血清型。RT-LAMP检测基于DNA合成过程中的等温自循环链置换。该方法的高特异性在很大程度上依赖于为扩增目标区域设计的引物组。我们设计了针对外壳蛋白基因内一个区域的特异性引物组，该区域包含O和N外壳蛋白类型典型的核苷酸特征，且这些引物的退火温度不同。将该检测方法与磁捕获总RNA提取相结合，我们建立了一种高度灵敏、简化且缩短的RT-LAMP程序，作为传统核酸检测诊断的替代方法。这种优化的病毒检测程序可在需要特定毒株时，在投入时间和精力进行多重RT-PCR检测之前，用作鉴定病毒血清型的初步测试。
