Using in silico analysis to investigate the false positive potential of qPCR systems for potato disease diagnosis

IF 1.7 3区农林科学 Q2 AGRONOMY

European Journal of Plant Pathology Pub Date : 2024-08-02 DOI:10.1007/s10658-024-02928-8

Junye Jiang, Will Feindel, Michael Harding, David Feindel, Stacey Bajema, Jie Feng

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Abstract

Potato (Solanum tuberosum) is one of the most important crops facing threats from different diseases. Rapid and accurate diagnosis is essential to control disease development and spread. Quantitative real-time PCR (qRT-PCR) has been widely used in potato disease diagnosis. In this study, we evaluated the specificity of 19 probe-based and four SYBR Green-based qPCR protocols for 17 potato diseases using in silico analysis. Primers and probes of those protocols were subjected to BLASTn analysis against the nucleotide collection (nr/nt) database and the whole-genome shotgun contigs (wgs) database of NCBI for the presence of primer/probe sequences in non-target species. Results showed that 12 of 23 qPCR protocols were not specific to the target pathogens. A qPCR experiment indicated that even nine single nucleotide polymorphisms (SNPs) are present on the sequences of the primer/probe binding sites between the potato silver scurf pathogen Helminthosporium solani and its close-related species H. velutinum, the primers/probe specific to the former could amplify signals from the latter. These findings highlight the need for additional methods to enhance the diagnostic accuracy and new sequencing technologies such as next generation sequencing could provide useful information to develop specific diagnostic protocols for these pathogens.

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利用硅分析研究用于马铃薯疾病诊断的 qPCR 系统的假阳性潜力

马铃薯（Solanum tuberosum）是面临不同病害威胁的最重要作物之一。快速准确的诊断对于控制病害的发展和传播至关重要。定量实时 PCR（qRT-PCR）已广泛应用于马铃薯病害诊断。在本研究中，我们利用硅分析评估了 19 种基于探针的 qPCR 方案和 4 种基于 SYBR Green 的 qPCR 方案对 17 种马铃薯病害的特异性。对这些方案的引物和探针进行了 BLASTn 分析，与 NCBI 的核苷酸集合（nr/nt）数据库和全基因组散射等位基因（wgs）数据库进行比对，以确定是否存在非目标物种的引物/探针序列。结果显示，23 个 qPCR 方案中有 12 个对目标病原体不具有特异性。一项 qPCR 实验表明，即使在马铃薯银屑病病原体 Helminthosporium solani 与其近缘种 H. velutinum 之间的引物/探针结合位点序列上存在 9 个单核苷酸多态性 (SNP)，前者的特异性引物/探针也能扩增后者的信号。这些发现突出表明，需要更多的方法来提高诊断的准确性，而新的测序技术（如新一代测序技术）可提供有用的信息，为这些病原体制定特定的诊断方案。

本文章由计算机程序翻译，如有差异，请以英文原文为准。

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来源期刊

European Journal of Plant Pathology 农林科学-农艺学

CiteScore

4.20

自引率

5.60%

发文量

183

审稿时长

3 months

期刊介绍： The European Journal of Plant Pathology is an international journal publishing original articles in English dealing with fundamental and applied aspects of plant pathology; considering disease in agricultural and horticultural crops, forestry, and in natural plant populations. The types of articles published are :Original Research at the molecular, physiological, whole-plant and population levels; Mini-reviews on topics which are timely and of global rather than national or regional significance; Short Communications for important research findings that can be presented in an abbreviated format; and Letters-to-the-Editor, where these raise issues related to articles previously published in the journal. Submissions relating to disease vector biology and integrated crop protection are welcome. However, routine screenings of plant protection products, varietal trials for disease resistance, and biological control agents are not published in the journal unless framed in the context of strategic approaches to disease management.