Austrian Agency for Health and Food Safety, Institute for Sustainable Plant Protection, 1220, Vienna, Austria.
University of Natural Resources and Life Sciences, Institute of Plant Protection, 3430, Tulln an der Donau, Austria.
J Virol Methods. 2021 Jun;292:114123. doi: 10.1016/j.jviromet.2021.114123. Epub 2021 Mar 9.
Over the course of developing and applying a new real-time PCR assay for the detection of the newly described apple chlorotic fruit spot viroid (ACFSVd), slight modifications of the reverse transcription (RT) step were found to improve significantly the detection limit of the assay. To prove this hypothesis, three different one-step RT-qPCR kits for the detection of three plant viroids and three plant viruses were compared. The results showed both extension of the RT reaction time from 10 or 15 min-30 min or the increase in reaction temperature from 49 to 52 °C for the cDNA synthesis step results in a 10 times higher sensitivity for potato spindle tuber viroid and apple scar skin viroid one-step RT-qPCR assay and 45 higher sensitivity for ACFSVd one-step RT-qPCR assay. No variation in the detection limit was observed when the modifications were tested on tomato brown rugose fruit virus, plum pox virus and tomato ringspot virus assays. This finding is highly valuable for the investigation of viroids in general and could contribute to enhance sensitivity in their detection and to benefit regulatory outcomes for national plant protection organisations.
在开发和应用新的实时 PCR 检测方法来检测新描述的苹果褪绿斑点类病毒（ACFSVd）的过程中，我们发现反转录（RT）步骤的轻微修改可以显著提高该检测方法的检测限。为了证明这一假设，我们比较了三种用于检测三种植物类病毒和三种植物病毒的一步法 RT-qPCR 试剂盒。结果表明，将 RT 反应时间从 10 或 15 分钟延长至 30 分钟，或者将 cDNA 合成步骤的反应温度从 49°C 升高至 52°C，均可使马铃薯纺锤块茎类病毒和苹果疤皮类病毒一步法 RT-qPCR 检测的灵敏度提高 10 倍，ACFSVd 一步法 RT-qPCR 检测的灵敏度提高 45 倍。在对番茄褐色皱果病毒、李痘病毒和番茄环斑病毒检测方法进行测试时，未观察到检测限的变化。这一发现对一般类病毒的研究具有重要价值，并有助于提高它们的检测灵敏度，使国家植物保护组织受益。
