Abstract An isothermal-based reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay was developed for the detection of Potato virus A (PVA). The assay was optimized with respect to the selection of primer, optimization of isothermal temperature, and its incubation period. Visual detection of the LAMP reaction was also developed by adding SYBR gold nucleic acid staining dye, where the change in color from dark orange to fluorescent yellowish green indicates a positive reaction. The assay was examined for its specificity with other viruses-infecting potato and with the host genome and was found specific to PVA. The sensitivity of the assay was found equally sensitive to that of the RT-PCR assay. The optimized assay was validated for the detection of PVA from potato leaf samples collected randomly from fields. The assay was also successful in the detection of PVA from potato tubers. To determine the virus-carrying aphids, a Squash Print RT-LAMP (SP-RT-LAMP) assay was developed. The SP-RT-LAMP assay was successful in the detection of PVA in single aphids. It was further validated to detect the target virus in single aphid samples collected randomly from potato canopy. The assay revealed that 33 of 69 single aphids were carrying the virus. We concluded that the assay is simple and sensitive for the detection of PVA in potato leaf and tubers and also in insect vector aphids.

中文翻译：

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用于检测马铃薯和昆虫载体蚜虫中的马铃薯病毒 a 的逆转录环介导等温扩增的开发

摘要 开发了一种基于等温逆转录环介导的等温扩增 (RT-LAMP) 检测方法，用于检测马铃薯病毒 A (PVA)。该测定在引物的选择、等温温度的优化及其孵育期方面进行了优化。LAMP 反应的视觉检测也通过添加 SYBR 金核酸染色染料开发，其中颜色从深橙色变为荧光黄绿色表示阳性反应。检查了该测定对其他病毒感染马铃薯和宿主基因组的特异性，发现它对 PVA 具有特异性。发现该测定的灵敏度与 RT-PCR 测定的灵敏度相同。优化后的测定法可用于从田间随机采集的马铃薯叶样品中检测 PVA。该测定还成功地检测了马铃薯块茎中的 PVA。为了确定携带病毒的蚜虫，开发了 Squash Print RT-LAMP (SP-RT-LAMP) 检测方法。SP-RT-LAMP 检测成功地检测了单个蚜虫中的 PVA。进一步验证了在从马铃薯冠层随机采集的单个蚜虫样本中检测目标病毒。分析表明，69 只蚜虫中有 33 只携带病毒。我们得出的结论是，该测定法对于检测马铃薯叶和块茎以及昆虫载体蚜虫中的 PVA 来说既简单又灵敏。进一步验证了在从马铃薯冠层随机采集的单个蚜虫样本中检测目标病毒。分析表明，69 只蚜虫中有 33 只携带病毒。我们得出的结论是，该测定法对于检测马铃薯叶和块茎以及昆虫载体蚜虫中的 PVA 来说既简单又灵敏。进一步验证了在从马铃薯冠层随机采集的单个蚜虫样本中检测目标病毒。分析表明，69 只蚜虫中有 33 只携带病毒。我们得出的结论是，该测定法对于检测马铃薯叶和块茎以及昆虫载体蚜虫中的 PVA 来说既简单又灵敏。