Kumar Ravinder, Kaundal Priyanka, Tiwari Rahul Kumar, Siddappa Sundaresha, Kumari Hema, Chandra Naga Kailash, Sharma Sanjeev, Kumar Manoj
ICAR-Central Potato Research Institute, Shimla, 171 001, H. P, India.
ICAR-Central Potato Research Institute, Regional Station, Modipuram, 250110, Uttar Pradesh, India.
Mol Cell Probes. 2021 Aug;58:101743. doi: 10.1016/j.mcp.2021.101743. Epub 2021 May 27.
Potato virus X (PVX), is a serious threat to global potato production. A simple and rapid detection method is imperative for PVX diagnosis and early management. In this study, an isothermal one-step reverse transcription-recombinase polymerase amplification (RT-RPA) method was optimized for the quick and convenient detection of PVX in potato leaves and tubers. Our results revealed that this one-step RT-RPA method was highly efficient than the conventional reverse transcription-polymerase chain reaction (RT-PCR). The amplification reaction was free from cross-reactivity with other common potato viruses and completed within 30 min. Moreover, this RT-RPA assay did not require a thermocycler based specific temperature phase amplification and can be easily performed using a simple heating block or water bath at a temperature range of 39-42 °C. The sensitivity assay demonstrated that the developed one-step RT-RPA method was 100 times more sensitive than a routine one-step RT-PCR. Initially, the purified total RNA as the template isolated from infected leaves of potato was used for the detection of PVX. One-step RT-RPA was later performed using cellular disc paper-based simple RNA extract as a template that could detect the virus more efficiently than purified total RNA. The performance of the one-step RT-RPA assay was further evaluated using 500 field samples of leaves and tubers representing different cultivars and geographical regions. To our knowledge, this is the first report of rapid, sensitive, and reliable detection of PVX infection by one-step RT-RPA using cellular disc paper-based simple RNA extract from leaves and dormant tubers of potato. It is superior to the common RT-PCR assay in terms of its versatility, quickness, and independence of highly purified RNA template and can be adopted as a substitute to RT-PCR as an effective technique for seed potato certification, quarantine, breeding, and field surveys.
马铃薯X病毒（PVX）对全球马铃薯生产构成严重威胁。一种简单快速的检测方法对于PVX诊断和早期管理至关重要。在本研究中，优化了一种等温一步逆转录重组酶聚合酶扩增（RT-RPA）方法，用于快速便捷地检测马铃薯叶片和块茎中的PVX。我们的结果表明，这种一步RT-RPA方法比传统的逆转录聚合酶链反应（RT-PCR）效率更高。扩增反应与其他常见马铃薯病毒无交叉反应，且在30分钟内完成。此外，这种RT-RPA检测不需要基于热循环仪的特定温度阶段扩增，在39-42°C的温度范围内使用简单的加热块或水浴即可轻松进行。灵敏度分析表明，所开发的一步RT-RPA方法比常规一步RT-PCR灵敏100倍。最初，以从感染马铃薯叶片中分离的纯化总RNA为模板检测PVX。后来使用基于细胞盘纸的简单RNA提取物作为模板进行一步RT-RPA，其检测病毒的效率比纯化总RNA更高。使用代表不同品种和地理区域的500份叶片和块茎田间样本进一步评估了一步RT-RPA检测的性能。据我们所知，这是首次报道使用基于细胞盘纸的马铃薯叶片和休眠块茎简单RNA提取物通过一步RT-RPA快速、灵敏且可靠地检测PVX感染。在通用性、快速性以及对高纯度RNA模板的独立性方面，它优于普通RT-PCR检测，可作为RT-PCR的替代方法，作为种薯认证、检疫、育种和田间调查的有效技术。
