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首页> 外文期刊>European Journal of Plant Pathology >Detecting potato viruses using direct reverse transcription quantitative PCR (DiRT-qPCR) without RNA purification: an alternative to DAS-ELISA
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Detecting potato viruses using direct reverse transcription quantitative PCR (DiRT-qPCR) without RNA purification: an alternative to DAS-ELISA

机译：使用直接逆转录定量PCR（DIRT-QPCR）检测马铃薯病毒而没有RNA纯化：Das-ELISA的替代方案

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摘要

Virus screening is obligatory to avoid the spread of plant viruses regionally and globally. Double-antibody sandwich (DAS)-ELISA is the standard for screening potato viruses owing to its high-throughput potential, robustness, and cost-benefit ratio. However, low virus titers present in dormant potato tubers may not be reliably detected by using DAS-ELISA. Virus enrichment for reliable virus detection by DAS-ELISA assay is time-consuming and can be avoided by switching to more sensitive molecular biological techniques. Therefore, we developed a TaqManA (R) qPCR-based one-step protocol, termed direct reverse transcription quantitative PCR (DiRT-qPCR) for detection of RNA potato viruses PVY, PLRV and PVS without sophisticated nucleic acid purification and providing a high-throughput potential. Compared with DAS-ELISA, DiRT-qPCR showed up to a 100,000,000-fold higher sensitivity depending on the virus species. We also compared the qualitative results of standard DAS-ELISA used in seed potato certification, performed by sampling leaves of at least 4-weeks-old cultivated tuber eye cuttings, to the 1.5 h long DiRT-qPCR protocol on dormant tubers. The DiRT-qPCR protocol achieved an agreement with the DAS-ELISA procedure of 92.8%, 84.1% and 82.3% for the detection of PLRV, PVY, and PVS, respectively. The investigated different virus species show different multiplication behavior in secondary infected potato tuber eye cuttings, which is assumed to be a reason for the remaining qualitative differences in the outcome of the DiRT-qPCR and DAS-ELISA comparison. In our opinion, DiRT-qPCR protocol can be used as a reliable, work- and resource-saving alternative to DAS-ELISA in qualitative directed virus detection, particularly because no RNA purification is needed and dormant potato tubers can be directly used.
机译：病毒筛查是避免在区域和全球范围内避免植物病毒的蔓延的义务。双抗体夹层（DAS）-Elisa是由于其高通量潜力，鲁棒性和成本效益比筛选马铃薯病毒的标准。然而，可以通过使用Das-ELISA可靠地检测到休眠马铃薯块茎中的低病毒滴度。 DAS-ELISA测定的可靠病毒检测病毒富集是耗时的，通过切换到更敏感的分子生物技术可以避免。因此，我们开发了一种基于TaqMana（R）QPCR的一步方案，称为直接逆转录定量PCR（DIRT-QPCR），用于检测RNA马铃薯病毒PVY，PLRV和PVS，没有复杂的核酸纯化并提供高通量潜在的。与Das-ELISA相比，Dirt-QPCR根据病毒物种，敏感度高达100,000,000倍的敏感性。我们还将通过至少4周龄栽培块茎切割的叶片的叶片进行了比较了种子马铃薯认证的标准DAS-ELISA的定性结果。污垢 - QPCR协议分别达到92.8％，84.1％和82.3％的DAS-ELISA程序分别检测PLRV，PVY和PV。研究的不同病毒物种在次要感染的马铃薯块茎眼切屑中显示出不同的乘法行为，这被认为是污垢 - QPCR和DAS-ELISA比较结果剩余定性差异的原因。在我们看来，污垢 - QPCR协议可以用作定性定向病毒检测中的DAS-ELISA的可靠，工作和资源替代品，特别是因为不需要RNA净化，并且可以直接使用休眠薯片。

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