A squash print reverse transcription loop-mediated isothermal amplification amplification (SP-RT-LAMP) assay was developed for detection of Potato leafroll virus (PLRV) in a single aphid. Healthy aphids were given an acquisition feeding on potato plants infected with PLRV, and the acquired aphids were used for RNA isolation. The RT-LAMP assay was carried out using LAMP primers targeting the coat protein gene of PLRV. The amplified product was run on agarose gel; a typical ladder-like pattern in the acquired aphids indicated that the assay was able to detect PLRV from an individual aphid. The results of the RT-LAMP assay for the detection of PLRV in a single aphid were also visualized directly by adding fluorescent dye in LAMP amplified product. These results were confirmed by RT-PCR detection where an amplicon of expected size of 492 bp was observed in virus-acquired single aphids. The assay was optimized with respect to its isothermal amplification temperature and its incubation period. The technique was examined for its relative selectivity with other potato viruses transmitted by aphids where it did not show any cross-reactivity with the tested viruses indicating its specificity. With respect to its relative sensitivity, it was found to be equally sensitive as the existing RT-PCR assay. It was also found to be robust and highly reproducible as it was able to detect PLRV from known and unknown single aphid samples. The assay was successful in detection of PLRV in potato plants and tubers as well. Hence, we conclude that the assay is simple, sensitive and economical for detection of PLRV in single aphid and in potato plants and tubers.

中文翻译：

---

南瓜印反转录环介导等温扩增法检测单蚜虫和马铃薯中的马铃薯卷叶病毒

开发了一种南瓜印逆转录环介导的等温扩增扩增 (SP-RT-LAMP) 测定法，用于检测单个蚜虫中的马铃薯卷叶病毒 (PLRV)。健康的蚜虫以感染 PLRV 的马铃薯植株为食，获得的蚜虫用于 RNA 分离。RT-LAMP 检测是使用针对 PLRV 外壳蛋白基因的 LAMP 引物进行的。扩增产物在琼脂糖凝胶上电泳；获得的蚜虫中典型的梯形图案表明该测定能够检测到来自个体蚜虫的 PLRV。RT-LAMP 检测单个蚜虫 PLRV 的结果也通过在 LAMP 扩增产物中添加荧光染料直接可视化。这些结果通过 RT-PCR 检测得到证实，其中在病毒获得的单蚜虫中观察到预期大小为 492 bp 的扩增子。该测定在其等温扩增温度和其孵育时间方面进行了优化。检查了该技术与由蚜虫传播的其他马铃薯病毒的相对选择性，它与测试病毒没有任何交叉反应，表明其特异性。就其相对灵敏度而言，发现它与现有的 RT-PCR 测定法同样灵敏。由于它能够从已知和未知的单个蚜虫样本中检测到 PLRV，因此还发现它具有稳健性和高度可重复性。该测定也成功地检测了马铃薯植物和块茎中的 PLRV。因此，我们得出结论，该测定很简单，